Genes of an otitis media isolate of nontypeable Haemophilus influenzae

ABSTRACT

The invention relates to the polynucleotide sequence of a nontypeable stain of  Haemophilus influenzae  (NTHi) and polypeptides encoded by the polynucleotides and uses thereof. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear and/or the nasopharynx.

RELATED APPLICATIONS

The present application is a divisional of U.S. Application No. 13/187,989filed Jul. 21, 2011, which is a divisional of U.S. Application No. 12/860,332 filed Aug. 20, 2010, (U.S. Patent No. 7,998,490), which is a divisional of U.S. Application No. 12/646,424, filed Dec. 23, 2009 (U.S. Patent No. 7,816,086), which is a continuation of U.S. Application No. 11/770,447, filed Jun. 28, 2007 (U.S. Patent No. 7,638,282), which is a continuation of U.S. Application No. 10/795,159, filed Mar. 5, 2004 (U.S. Patent No. 7,241,867) which claims priority benefit from U.S. Provisional Application 60/453,134 filed Mar. 6, 2003 which is incorporated herein by reference in their entirety.

Part of the work during the development of this invention was made with government support from the National Institutes of Health under grant number R01DC03915. The U.S. Government has certain rights in the invention.

FIELD OF INVENTION

The invention relates to the polynucleotide sequence of a nontypeable strain of Haemophilus influenzae (NTHi) genome, NTHi genes contained within the genome and polypeptides encoded by the polynucleotides. The invention also relates to uses of these NTHi polynucleotides and NTHi polypeptides including vaccines and methods of treating and preventing NTHi related disorders. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear or nasopharynx.

BACKGROUND

Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez, Epidemiol. Rev., 15:444-465, 1993). Recent statistic indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980's. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child's behavior, education and development of language skills (Baldwin, Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162:685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis. J., 16: 59-11, 1997).

Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear. the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are 13%; Bright et al., Am. J. Public Health, 83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al., Pediatrics, 93(3):353-63, 1994; Bright et al., supra.; Cimons, ASM News, 60: 527-528; Paap, Ann. Pharmacother., 30(11): 1291-7, 1996).

Progress in vaccine development is most advanced for Streptococcus pneumoniae, the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al., Pedriatr. Infect. Dis J., 19: 187-195; Eskola et al., Pedriatr. Infect. Dis J., 19:S72-78, 2000; Eskola et al., N. Engl. J. Med. 344: 403-409, 2001; Snow et al., Otol. Neurotol., 23: 1-2, 2002). Less progress has been made for non-typeable Haemophilus influenzae (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein, Pedriatr. Infect. Dis J., 16: S5-8, 1997; Spinola et al., J. Infect. Dis., 154: 100-109, 1986). Hampering development of effective vaccines against NTHi, is the currently incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay is a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host's immune response as the disease progresses from one of host immunological tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space.

Currently there is a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank, Pedriatr. Infect. Dis J., 13(11): 1064-8, 1994: Karma et al., Int. J. Pedritr. Otorhinolaryngol., 32(SuppL): S127-34, 1995).

SUMMARY OF INVENTION

The present invention provides for the identification and characterization of the genomic sequence of NTHi H. influenzae strain 86-028NP and the polypeptide sequences encoded thereby. The 3-fold analysis of the NTHi genomic sequence is set out in a series of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent 8-fold analysis of the genomic sequence is set out in a series of 11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs are raw data and one of skill in the art may assemble these contigs by comparing overlapping sequences to construct the complete genome of the NTHi stain 86-028NP using routine methods.

The present invention also provides for antibodies specific for the NTHi polypeptides of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting NTHi polynucleotides with specific polynucleotide probes or detecting NTHi polypeptides with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria.

The present invention also contemplates methods of eliciting an immune response by administering a NTHi polypeptide of the invention or a NTHi peptide thereof. These methods include administering the NTHi polypeptide or NTHi peptide as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM. The following NTHi genes are upregulated during or in response to middle ear and/or nasopharynx infections; and the polypeptides encoded by these genes and peptides thereof are contemplates as possible OM vaccine candidates and/or target of chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, urcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064, NTHi hisB gene is set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as nucleotide sequence SEQ ID NO: 619 and encodes the amino acid sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as nucleotide sequence SEQ ID NO: 621 and encodes the amino acid sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as nucleotide sequence SEQ ID NO: 623 and encodes the amino acid sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as nucleotide sequence SEQ ID NO: 625 and encodes the amino acid sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as nucleotide sequence SEQ ID NO: 627 and encodes the amino acid sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as nucleotide sequence SEQ ID NO: 629 and encodes the amino acid sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as nucleotide sequence SEQ ID NO: 631 and encodes the amino acid sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as nucleotide sequence SEQ ID NO: 633 and encodes the amino acid sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as nucleotide sequence SEQ ID NO: 635 and encodes the amino acid sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as nucleotide sequence SEQ ID NO: 637 and encodes the amino acid sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as nucleotide sequence SEQ ID NO: 639 and encodes the amino acid sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as nucleotide sequence SEQ ID NO: 641 and encodes the amino acid sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as nucleotide sequence SEQ ID NO: 643 and encodes the amino acid sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as nucleotide sequence SEQ ID NO: 645 and encodes the amino acid sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as nucleotide sequence SEQ ID NO: 647 and encodes the amino acid sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as nucleotide sequence SEQ ID NO: 649 and encodes the amino acid sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as nucleotide sequence SEQ ID NO: 651 and encodes the amino acid sequence set out as SEQ ED NO: 652. NTHi HI1063 gene is set out as nucleotide sequence SEQ ID NO: 653 and encodes the amino acid sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as nucleotide sequence SEQ ID NO: 655 and encodes the amino acid sequence set out as SEQ ID NO: 656, NTHi HI1292 gene is set out as nucleotide sequence SEQ ID NO: 657 and encodes the amino acid sequence set out as SEQ ID NO: 658.

The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx, and therefore are contemplated to encode OM vaccine candidates and/or targets of chemotherapy. In addition, the following NTHi genes are contemplated to be virulence-associated genes and therefore are contemplated to encode possible OM vaccine candidates and/or targets of chemotherapy: HI1386, HI1462. HI1369, lav, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659 and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the amino acid sequence set out as SEQ ID NO: 662. NTHi Mil 369 gene sequence is set out as SEQ ID NO: 665 and encodes the ammo acid sequence set out as SEQ ID NO: 666. NTHi lav gene sequence is set out as SEQ ID NO: 663 and encodes the amino acid sequence set out as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes associated with virulence include the polynucleotide sequences presented as SEQ ID NO: 667 and SEQ ID NO: 673.

As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of the NTHi polypeptides, which are upregulated or active during infection. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating NTHi protein expression by administering an antisense oligonucleotide that specifically binds to NTHi genes that are upregulated during NTHi infections, such genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of the proteins encoded by theses genes. The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ FD NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx and therefore antisense oligonucleotides that specifically bind these polynucleotide sequences are also contemplated.

Polynucleotides and Polypeptides of the Invention

The present invention provides for the sequences of the NTHi strain 86-028NP genome. This genomic sequence is presented as a series of contig sequences denoted herein as “contigs 1-576”. Each contig is assigned a sequence identification number that correlates with its “contig number”. Therefore, the contigs of the present invention as set out as SEQ ID NOS: 1-576. These contig polynucleotide sequences may be assembled into the complete genome sequence of the NTHi strain 86-028NP using routine methods. Upon completion of 8-fold sequence analysis of the NTHi strain 82-028NP genome, the genomic sequence was assembled into 11 contigs which are denoted herein as SEQ ID NOS: 675-685.

The present invention provides for the NTHi polynucleotide sequences and open reading frames contained within the contigs of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5. The present invention also provides for the polypeptide sequences encoded by the NTHi polynucleotides of the present invention such as the amino acid sequences set out in Table 3B, Table 4B and Table 5. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotides sequence of SEQ ID NOS: 1-576; SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide which is an allelic variant of any polynucleotides recited above; (c) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (d) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention.

The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%. 96%, 97%, 98% or 99% sequence identity to the NTHi polynucleotides recited above.

Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to the NTHi nucleotide sequences of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, or compliments thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences.

The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 19.89). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.3% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.

The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, and nucleotide sequences out in Table 3B, Table 4B and Table 5 herein, preferably the open reading frames therein, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, fable 4B and Table 5 with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.

Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences encoded by the nucleotide sequences included within the polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or the corresponding full length or mature protein. The polypeptides of the invention include the ammo acid sequences of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622. SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644. SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662. SEQ ID NO: 664, SEQ ID NO: 666. SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695. SEQ ED NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741. SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753. SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771 which are set out in Table 3B, Table 4B and Table 5 herein.

Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) an open reading frame contained within the nucleotide sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.

The invention also provides biologically active or immunologically active variants of the amino acid sequences of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%. 89%, at least about 90%, 91%. 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides encoded by the polynucleotides included within the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having an amino acid sequence set out in Table 3B, Table 4B and Table 5 herein

NTHi peptides icier to fragments of the NTHi polypeptides encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 or the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein. The preferred NTHi peptides are biologically and/or immunologically active.

The present invention further provides isolated NTHi polypeptides or NTHi peptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORE) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.

The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions which may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a normative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic; arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine General rules for amino acid substitutions are set forth in Table 1 below.

TABLE 1 Amino Acid Substitutions Original Residues Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asn Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu Leu Norleucine, Ile, Val, Met, Leu Lys Arg, 1,4 Diaminobutyric Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Arg Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu

Antisense polynucleotides complementary to the polynucleotides encoding the NTHi polypeptides are also provided.

The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells comprising polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as E. coli. Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia.

The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs.

Antibodies and Methods for Eliciting an Immune Response

The invention provides antibodies which bind to antigenic epitopes unique to (i.e., are specific for) NTHi polypeptides. Also provided are antibodies which bind to antigenic epitopes common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and Ffab)2 fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art.

It is known in the art that antibodies to the capsular polysaccharide of H. influenzae exhibit the ability to kill bacteria in vitro assays. These antibodies are also known to protect against challenge with H. influenzae in animal model systems. These studies indicate antibody to the capsular polysaccharides are likely to elicit a protective immune response in humans. The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both H. influenzae bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention which reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration into the epithelium of H. influenzae bacteria or enhance phagocytosis of the H. influenzae bacteria.

In vitro complement mediated bactericidal assay systems (Musher et al., Infect, Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) may be used to measure the bactericidal activity of anti-NTHi antibodies. Further data on the ability of NTHi polypeptides and NTHi peptides to elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein.

It is also possible to confer short-term protection to a host by passive immunotherapy via the administration of pre-formed antibody against an epitope of NTHi, such as antibodies against NTHi OMP. LOS or noncapsular proteins. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against NTHi epitopes.

The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods include immune responses which kill the NTHi bacteria and immune responses which block H. influenzae attachment to cells. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention. In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing a NTHi protein or NTHi peptide of the invention. In yet another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding a NTHi protein or NTHi peptide of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual.

An “immunological dose” is a dose which is adequate to produce antibody and/or T cell immune response to protect said individual from NTHi infection, particularly NTHi infection of the middle ear and/or the nasopharynx or lower airway. Also provided are methods whereby such immunological response slows bacterial replication. A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the NTHi polypeptides encoded by the polynucleotide of the invention or antigenic peptides thereof.

The invention correspondingly provides compositions suitable for eliciting an immune response to NTHi infection, wherein the antibodies elicited block binding of NTHi bacterium to the host's cells. The compositions comprise NTHi proteins or NTHi peptides of the invention, cells expressing the NTHi polypeptide, or polynucleotides encoding the polypeptides. The compositions may also comprise other ingredients such as carriers and adjuvants.

Genes that are up-regulated in NTHi infection of the middle ear and/or the nasopharynx and genes that are associated with NTHi virulence are described herein. The polypeptides and peptides thereof which are encoded by these NTHi genes are contemplated to be useful for eliciting an immune response for treating or preventing disorders associated with NTHi infection, such as OM. Some of the polypeptides encoded by these genes include: histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, Phosphoribosylaminoimidazole carboxylase catalytic subunit, ornithine carbarnolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phosphocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein. MukF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, MukF, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665. HI1292, HI1064 HI186, HI0352 genes. NTHi OMPs, LOS and noncapsular proteins are also contemplated to elicit an immune response for prevention and treatment of disorders associated with NTHi infection.

An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic.

The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise administering antibodies or polypeptides of the invention that block binding of NTHi cellular attachment. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment.

Pharmaceutical compositions comprising antibodies of the invention, polypeptides of the invention and/or small molecules of the invention that block NTHi cellular attachment are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.

Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting NTHi polynucleotides of the invention in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotide may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR.

The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of NTHi antigens (NTHi polypeptides and peptides thereof) in various body fluids of individuals suspected of H. influenzae infection. In another embodiment, the NTHi proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and Immunoelectrophoresis assays.

Vaccines and Chemotherapeutic Targets

An aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a NTHi antigen protein or an antigenic peptide thereof.

The present invention also provides for vaccine formulations which comprise an immunogenic recombinant NTHi protein or NTHi peptide of the invention together with a suitable carrier. The NTHi polypeptides and peptides thereof contemplated as vaccine candidates and/or targets of chemotherapy include, but are not limited to, histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate synthase, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MUKF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, Had A and those hypothetical proteins encoded by HI10094, HI1163, HI0665, HI1292, HI1064 HI1386, HI0352 genes, NTHi OMPs, NTHi LOS and NTHi noncapsular proteins and polypeptides encoded by the novel NTHi polynucleotide sequences present in the nucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Fable 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein.

Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

A. Peptide Vaccines

Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal or immunized human.

The NTHi antigenic peptides of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v. 2, Academic Press, New-York, p. 255). Automated solid phase synthesizers are commercially available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTHi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al., J Am Chem Soc., 103: 3185 (1981).

Some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid and the like, and coupling agent which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carobxylate (SMCC).

B. Vaccine Compositions and Administration

A priming dose of the immunogen that is followed by one or more booster exposures to the immunogen may be necessary to be an effective vaccine (Kramp et al., Inject. Immun., 25: 771-773, 1979: Davis et al., Immunology Letters, 14: 341-8 1986 1987). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immune-modulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.

The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include emulsions such as Freund's Adjuvants and other oil emulsions. Bordetella pertussis, MF59, purified saponin from Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from E. coli, holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages.

Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.

Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine or amphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, dioleoyl phosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark (Quillaja saponaria) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery.

The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration.

Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing the NTHi polypeptide or NTHi peptides of the invention are administered to a patient susceptible to or otherwise at risk of bacterial infection to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. Such an amount is defined to be an “immunogenically effective dose.” In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70 kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi polypeptide or NTHi peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.

Humoral immune response may be measured by many well known methods, such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI). In particular. SRID utilizes a layer of a gel, such as agarose, containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested, EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilution of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.

Nontypeable Haemophilus influenzae (NTHi)

H. influenzae is a small, nonmotile gram negative bacterium. Unlike other H. influenzae strains, the nontypeable H. influenzae (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b H. influenzae isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins and noncapsular proteins.

Humans are the only host for H. influenze. NTHi strains commonly reside in the upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTHi causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis. Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis.

Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et al., Infect. Immun., 36; 535-40, 1982), enzyme allotypes (Musser et al., Infect. Immun., 52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contains two common OMP's with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP's within the 32,000-42,000 dalton range. The NTHi liposaccharide profile is fundamentally different than the enteric gram negative bacteria and separates into 1-4 distinct bands ranging from less than 20,000 daltons.

A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Immun., 62: 1710-8, 1994: DeMaria et al., Infect. Immun., 64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear.

DFI Strategy

A differential fluorescence induction (DFI) strategy was used herein to identify NTHi genes induced during OM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; signature-tagged mutagenesis (STM) enabling tag-specific identification of genes that alter the virulence properties of a microorganism when mutated; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses Fluorescent Activated Cell Sorting (FACS) FACS analysis to select for transcriptionally active promoters (Chiang et al., Annu. Rev. Microbiol., 53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro .

DFI has been successfully utilized in many microorganisms. For example, a Green Fluorescent Protein (GFP) GFP reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al., Mol. Microbiol., 17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when Salmonellae were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow, Mol. Microbiol., 22: 367-378, 1996; Valdivia and Falkow, Science, 277: 2007-2011, 1997; Valdivia and Falkow, Curr. Opin. Microbiol., 1: 359-363, 1998). In addition, DFI has been used to identify promoters expressed in S. pneumoniae and S. aureus when grown under varied in vitro conditions simulating infection (Marra et al., Infect. Immun., 148: 1483-1491, 2002 70(3): 1422-1433, 2002; Schneider et al., Proc. Natl. Acad. Sci. U.S.A., 97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in Bacillus cereus in response to environmental stimuli (Dunn and Handelsman, Gene, 226: 297-305, 1999), in S. pneumoniae in response to a competence stimulatory peptide (Bartilson et al., Mol. Microbiol., 39: 126-135, 2001), and upon interaction with and invasion of host cells in Bartonella henselae Lee and Falkow, Infect. Immun., 66: 3964-3967, 1998), Listeria monocytogenes Wilson et al., Infect. Immun., 69: 5016-5024, 2001), Brucella abortus (Eskra et al., Infect. Immun., 69: 7736-7742, 2001), and Escherichia coli (Badger et al., Mol. Microbiol., 36: 174-182, 2000).

Whereas DFI has been successfully used to identify promoters active in cell culture models of infection or in vitro conditions designed to simulate an in vivo environment, few have applied DFI to identify promoters regulated in a specific biological niche within the whole animal. This is likely due to the numerous challenges associated with sorting from an in vivo environment. The host inflammatory response, dissemination and/or clearance of bacterial cells from the site of infection, as well as adherence of bacteria to epithelial cells, possibly via biofilm formation, can make bacteria inaccessible for retrieval from the living animal. These factors, among others, contribute to the complexity of the microenvironment and the heterogeneity of gene expression as the bacteria sense and respond to these changes. Recently, DFI has been used to identify promoters expressed in S. pneumoniae when the bacteria were screened in a mouse mode) of respiratory tract infection and a gerbil infection model of OM (Marra et al., Infect. Immun. 70: 1422-33, 2002; Marra et al., Microbiol., 148: 1483-91, 2002).

Animal Model

The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol. 4: 223-225, 1997; Suzuki and Bakaletz, Infect. Immun., 62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al., Vaccine, 15: 955-961, 1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).

In particular, there is an unique in vivo model wherein adenovirus predisposes chinchillas to H. influenzae-induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993: Suzuki et al., Infect Immunity 62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4): 2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).

Genes Unregulated In vivo in Response to NTHi Infection of the Middle Ear

In order to identify differentially regulated promoters in response to NTHi infection of the middle ear, a promoter trap library was constructed and sorting parameters defined. A portion of the promoter trap library was inoculated directly into the chinchilla middle ear and OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. In addition, the middle ear fluids were recovered 24 and 48 hours after infection. Two-color FACS analysis was used to isolated bacteria that were expressing GFP from other cells and debris associated with the effusion. Following isolation, DNA sequence of the Haemophilus inserts 5′ of the gfpmut3 gene were determined and analyzed, in this manner, we identified genes that are up-regulated as NTHi sense and respond to the environment of the chinchilla middle ear during AOM. The following genes were identified and due to their up-regulation during NTHi infection, they may play a role in NTHi infection and virulence.

As described below in Example 7, following the DFI procedure described above and subsequent FACS analysis of gfp-expressing clones, 52 candidate clones containing potential in vivo-regulated promoters were isolated. The genes these clones control were categorized based upon general description and function within the cell and include general metabolic processes, environmental informational processing and membrane transport, membrane proteins and hypothetical proteins. Eight of these 52 clones contain sequences that are unique to NTHi strain 86-028NP. Importantly, 3 clones were isolated from independent screens in more than one animal thereby verifying the method of isolation.

In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) RT-PCR. The parent strain 86-028NP, was used for these studies. Thus, wild-type gene expression without the influence of plasmid copy number on gene regulation was analyzed, allowing for the indication of false-positive clone identification by FACS. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear. This analysis identified in vivo-regulated promoters which drive expression of genes involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. (See Table 4 in Example 6).

Quantitative RT-PCR demonstrated a two-fold increase in lolA expression, enabling lipoprotein transport from the inner membrane to the outer membrane. Bacteria grow rapidly in the middle ear environment reaching 5.0×10⁸ CFU NTHi ml middle ear fluid within 48 hours. The bacteria sense and respond to the environment, acquiring or synthesizing the necessary nutrients for growth and survival. The gene encoding the membrane component in ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5 fold increase in expression in vivo compared to cells growing in vitro. In addition, many genes involved in metabolic processes show a dramatic increase in gene expression in vivo compared to cells growing in vitro. These include a riboflavin synthesis gene, ribB (SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625), involved in arginine degradation via the urea cycle and uxuA (SEQ ID NO: 627), encoding mannonate hydrolase, required for the uptake of D-glucuronate and transformation into glyceraldehyde 3 phosphate. In addition, but to a lesser degree, genes for histidine biosynthesis (hisB; SEQ ID NO: 615). DNA repair (radC; SEQ ID NO: 639) and a putative intracellular septation transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated.

Disulfide bond formation is important for folding and assembly of many secreted proteins in bacteria. In prokaryotes, DsbA and DsbB make up the oxidative pathway responsible for the formation of disulfides. DsbB reoxidizes DsbA, which donates disulfide bonds directly to unfolded polypeptides, and DsbB has been demonstrated to generate disulfides de novo from oxidized quinones (Collet and Bardwell, Mol. Microbiol., 44: 1-8, 2002). In H. influenzae strain Rd, DsbA is required for competence for transformation (Tomb, Proc. Natl. Acad. Sci. U.S.A., 89: 10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB gene (SEQ ID NO: 629) transcription was demonstrated, illuminating an important role for disulfide interchange for NTHi growing in the middle ear environment.

Bacteria colonization of the middle ear, a normally sterile environment, results in a host inflammatory response and subsequent neutrophil infiltration. Bacteria have evolved numerous strategies to combat this host response. NTHi increase gene expression (4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required for expression of active urease in Helicobacter, shown to be involved in acid tolerance (Young et al., J. Bacterol., 178: 6487-6495, 1996). Recently, it has been reported that urease activity may play a role in chronic Actinobacillus pleuropneumoniae infection by counteracting the decrease in pH occurring upon infection (Baltes et al., Infect. Immun., 69: 472-478, 2000; Baltes et al., Infect. Immun., 69; 472-478, 2001; Bosse and Machines, Can. J. Vet. Res., 64: 145-150). A biotype analysis on NTHi isolates from middle ear effusions demonstrated that 87% are urease positive (DeMaria et al., J. Clin. Microbiol., 20:1102-1104, 1984). However, the role of urease in NTHi virulence is unknown. Similarly, an increase in expression of a gene whose product demonstrates 88% sequence identity to a pyridoxine biosynthesis protein in S. pneumoniae and 60% homology to a putative singlet oxygen resistance protein that may function as an antioxidant. Phosphorylcholine (ChoP) has been implicated in the pathogenesis of NTHi (Weiser et al., Inject. Immun., 65: 943-950, 1997). NTHi modulates ChoP expression by phase variation, decorating the LOS on the cell surface. ChoP may contribute to NTHi persistence in the respiratory tract via decreased susceptibility to antimicrobial peptides (Lysecko et al., Infect. Immun., 68: 1664-1671, 2000) and alter the sensitivity to serum killing mediated by C reactive protein (CRP) (Weiser et al., J. Exp. Med., 187: 631-640, 1998). The microenvironment of the nasopharynx and middle ear cavity may select for the ChoP⁺ phenotype, as ChoP⁺ strains show greater colonization of the chinchilla nasopharynx (Tong et al., Infect. Immun., 68: 4593-4597, 2000). Expression of the licC gene (SEQ ID NO: 633) was also increased. The licC gene encodes a phosphorylcholine cytidylyltransferase that plays a role in the biosynthesis of phosphorylcholine-derivatized LOS (Rock et al., J. Bacterol., 183: 4927-4931, 2001).

Also included among the in vivo-induced genes is a set whose products subsequently regulate gene expression or DNA replication. These genes include transcriptional regulation of glycerol metabolism by the glp repressor, glpR (SEQ ID NO: 643), the arginine repressor gene, argR (SEQ ID NO: 647), and the integration host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a histone-like protein that binds DNA at specific sequences, an accessory factor involved in replication, site-specific recombination and transcription, altering the activity of a large number of operons (Goosen and van de Putte, Mol. Microbiol. 16:1-7, 1995). In addition, CspD inhibits DNA replication during stationary phase-induced stress response in E. coli (Yamanaka et al., Mol. Microbiol., 39: 1572-1584, 2001) and the mukF (SEQ ID NO: 641) gene protein homologue contributes to a remodeling of the nucleiod structure into a more compact form prior to cell segregation (Sawitzke and Austin, Proc. Natl. Acad. Sci. U.S.A., 62: 1710-1718, 2000). The DFI strategy described herein also identified promoters induced in vivo for genes of unknown function. The hypothetical protein, HI0094, demonstrated an 8-fold increase in gene expression during early OM but its role remains unknown HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli.

A high-density transposon mutagenesis strategy was used to identify H. influenzae genes essential for growth on rich medium (Akerley et al., Proc. Natl. Acad. Sci. U.S.A., 99: 966-971, 2002). Six genes were identified in the screen described herein that are included in essential gene set described in Akerley et al., supra. (hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes of non-typeable H. influenzae that are expressed upon interaction with two human respiratory tract-derived epithelial cell lines have been identified. These genes included those involved in metabolic processes, stress responses, gene expression, cell envelope biosynthesis, DNA-related processes, cell division and ORE's encoding proteins of unknown function. (Ulsen et al., Mol. Microbiol., 45: 485-500, 2002). Similarly the stress response gene, cspD (SEQ ID NO: 649), genes involved in purine and riboflavin biosynthesis, and a protein of unknown function, vapA was identified in the screen described herein. Expression of vapA was detected in vitro, yet vapA gene expression increased two-fold in vivo. These unique approaches identified known genes that are upregulated in NTHi-induced OM and therefore are likely to play a role in NTHi infection and virulence; and may be potential candidates for vaccines and antisense therapies and other therapeutic methods of treatment of NTHi related disorders.

The DFI strategy resulted in the identification of promoters induced in vivo for genes of unknown function as well. The hypothetical protein, HI0094, demonstrated a 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli. Therefore, these hypothetical genes are likely to play a role in OM induced by NTHi infection.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the LKP gene region in a panel of Haemophilus isolates. The strain 86-028NP sequence is identical in this region to the sequence in NTHi strain R3001. Both of these NTHi lack the h if gene cluster encoding the hemagglutinating pilus.

FIG. 2 depicts the rfaD region in a panel of Haemophilus isolates. The gene arrangement in the rfaD region of the strain 86-028NP genome is similar to that of the strain Rd genome but different than the arrangement of these genes seen in the genome of most NTHi examined.

FIGS. 3A-3M set out the nucleotide sequences (SEQ ID NOS: 589-614) described in fable 4, which were identified to be upregulated during OM infection (see Example 6). The nucleotides (nt.) which correspond to known genes and those nt. which correspond to the contig sequences set out as SEQ ID NO: 1-576 are also presented.

DETAILED DESCRIPTION

The following examples illustrate the invention wherein Example 1 describes the sequence of the NTHi genome, Example 2 describes the identified contigs and initial gene discovery, Example 3 describes construction of the NTHi promoter trap library, Example 4 describes the analyses of 86-028NP derivatives expressing GFP, Example 5 demonstrates direct labelling of bacteria from middle ear fluids, Example 6 describes identification of promoters induced in vivo in acute otitis media, Example 7 describes identification of virulence-associated genes, and Example 8 describes identification of unique NTHi gene sequences.

Example 1 Sequence of a Non-Typeable Haemophilus influenzae Genome

NTHi strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM at Columbus Children's Hospital. (Bakaletz et al. Infection and Immunity, 56(2): 331-335, 1988) The 86-028NP strain was deposited with the American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct. 16, 2002 and assigned accession no. PTA-4764.

In an effort to more broadly approach the identification of the virulence determinants in NTHi, the genome of the NTHi 86-028NP strain was sequenced to 3-fold coverage. Chromosomal DNA was prepared from strain 86-028NP using the Puregene protocol and sheared to 2-4 kb in size with a Hydroshear instrument (Gene Machines). The sheared DNA was ethanol-precipitated, end-repaired using a mixture of Klenow enzyme and T4 DNA polymerase, and size-selected by agarose gel electrophoresis to obtain 2-4 kb fragments as described in Chissoe et al. (Methods: a Companion to Methods of Enzymology 3: 55-65, 1991) and Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2^(nd) Ed. Cold Springs Harbor, N.Y., 1989). These fragments were cloned into vector pUC18 using the SmaI restriction site (phosphatase-treated) and transformed into E. coli XL-1 Blue, selecting for ampicillin resistance. Colonies that contain inserts were identified by blue/white screening on LB-Amp plates containing X-gal, and transferred into 96-deep well plates containing 1.5 ml of TB-Amp (TB=Terrific Broth) broth. The deep-well plate cultures were grown overnight (18-22 hours) at 37° C. Template preparation, sequencing and contig assembly were performed.

Automated template preparation was performed on the Beckman Biomek 2000 automated robotics workstation as described in Chissoe et al., (supra.) Briefly, each 96-deep well plate, containing the clones prepared above, was centrifuged to pellet the cells, the supernatant decanted, and the cells frozen (if necessary) at −20° C. Four 96-deep well blocks were placed on the Biomek table, and the liquid handling robot was used to prepare the template using an automated version of a typical SDS-NaOH lysis protocol as described in Chissoe et al., (supra.). The final ethanol-precipitated templates were each dissolved in 50 μl ddH₂O, and used for DNA sequencing.

Sequencing reactions were run by re-arraying the templates (from 96-well plates) into 384-well plates, using the Robbins Hydra 96 robot. Cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse), cleaned up over Sephadex G50 columns, and analyzed on a PE Biosystems 3700 capillary electrophoresis DNA sequencer according to the manufacturer's instructions. Sequencing reads (8219) were assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The statistics for the 3-fold sequencing are shown in Table 2A. The total unique sequence in assembly 17 is 1.74 Mb.

TABLE 2A Contig Size Total Number Total Length % of Cumulative 0-1 kb 65 55961 3.2% 1-2 kb 228 333665 19.2% 2-3 kb 101 243059 14.0% 3-4 kb 49 172385 9.9% 4-5 kb 45 196699 11.3% 5-10 kb  74 515152 29.6% 10-20 kb  11 144591 8.3% 20-30 kb  3 77352 4.4%

Subsequently, 8-fold sequencing analysis of the NTHi genome was carried out. The 8-fold sequencing assembled the NTHi genome into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID NOS: 675-685 herein. The statistics for the 8-fold sequencing are shown in Table 1B.

TABLE 2B Contig Size Total Number Total Length % of Cumulative 0-1 kb 5 3950 0.2% 1-2 kb 3 4316 0.2% 2-3 kb 0 0 0.0% 3-4 kb 1 3964 0.2% 4-5 kb 0 0 0.0% 5-10 kb 0 0 0.0% 10-20 kb 1 15147 0.8% 20-30 kb 2 51888 2.7% 30-40 kb 0 0 0.0% 40-50 kb 0 0 0.0% 50-100 kb 1 85814 4.5% >100 kb 5 1760339 91.4%

Example 2 Contig Description and Initial Gene Discovery

Seventy-five of the 88 contigs with length ≧5000 bp, identified with the 3-fold sequence analysis, show significant similarity via BLASTN to genes in H. influenzae strain Rd. To visualize the potential relationship between the gene order in H. influenzae strain 86-028NP and H. influenzae strain Rd, the 86-028NP three-fold contig set and the Rd gene set were bidirectionally compared using BLASTN. The results were plotted in gene-order verses contig space by sorting the contigs based on gene coordinates of the Rd genes hit, anchoring each contig at the smallest coordinate found as described in Ray et al., (Bioinformatics 17: 1105-12, 2001). Compared in this fashion, an incomplete assembly of a genome with identical gene order to a completely known genome would display a monotonically increasing stair-stepped form.

BLASTX was used to identify hits to sequences with homology to genes in the strain Rd genome as well as genes not found in H. influenzae strain Rd. Hits to strain Rd sequences were removed from the data set and the other hits summarized in Table 3A. The data are presented as follows: contig # (=SEQ ID NO: #), column 1; E score for each hit, column 2; the name of the protein that had homology to a portion of the amino acid translation of the cited contig, column 3; the organism producing the homologue, column 4; and the Genbank protein identifier for each of the proteins cited in column 3, column 5; the corresponding nucleotides within the contig (referenced by SEQ ID NO). In most instances, several homologues were identified but for clarity, the protein of greatest homology is cited in Table 3A.

The sequences for some of the genes listed in Table 3A were identified within the 8-fold sequencing of the NTHi genome. Table 3B lists the location of these genes within the 11 contigs, the full length open reading frame sequence (identified by SEQ ID NO:), the derived amino acid sequence encoded by the open reading frame and the gene with high homology identified by BLASTX (as listed in Table 3A).

To examine the relative short range gene arrangements in NTHi and the Rd strain, the gene order in two gene clusters that have been well-described were compared. First, the genes present in the hemagglutinating pilus (LKP) gene region were examined. (Mhlanga-Mutangadura et al., J Bacterial. 180(17): 4693-703, 1998). The pilus gene cluster is located between the purE and pepN genes, only fragments of which are depicted in FIG. 1. The serotype b strain, Eagan, contains the hifABCDE gene cluster and produces hemagglutinating pili. Strain Rd lacks the hicAB genes as well as the hifABCDE gene cluster. In general, the nontypeable strains previously examined contained the hicAB genes but not the hif genes that encode the hemagglutinating pilus. The strain 86-028SNP sequence (described herein) is identical in this region to the sequence in NTHi strain R3001 (FIG. 1). The rfaD gene encodes an enzyme involved in the biosynthesis of endotoxin. In addition, the rfaD gene from NTHi strain 2019 has been characterized by Nichols et al. (Infect Immunity 65(4): 1377-86, 1997). In strain 2019, the rfaD gene is immediately upstream of the rfaF gene that encodes another enzyme involved in endotoxin biosynthesis. The gene arrangement in strain Rd is different; the rfaD and rfaF genes are separated by approximately 11 kb of sequence. Most nontypeable strains examined contained the gene arrangement seen in strain 2019. In contrast, strain 86-028NP has a gene arrangement identical to that seen in strain Rd (FIG. 2).

A global analysis of the current assembly indicates that the gene content and order are similar to that in strain Rd. A more detailed analysis revealed that there are a substantial number of NTHi genes not previously seen in the Pasteurellaceae and some regions where the NTHi gene content and order is different than that seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features.

The DFI strategy also identified novel NTHi sequences that had increased gene expression. A list of these novel contig sequences that contain genes or gene fragments that have homology to ORFs in other organisms (primarily gram-negative bacteria) is set out in Table 3A. For example, the nucleotide sequence of contig 442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous to the sequences encoding amino acids 1-116 of H. influenzae strain Rd lipoprotein B (LppB). The gene is positioned between the stationary phase survival gene, surE, and a gene encoding a 43 kD antigenic outer membrane lipoprotein that is highly homologous to the recently identified bacterial lipoprotein, LppB/NlpD, which has been associated with virulence (Padmalayam et al., Infect. Immun., 68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated that nlpD and surE gene expression was induced during stationary phase of bacterial growth in Thermotoga maritima (Zhang et al., Structure (Camb), 9: 1095-1106, 2001). Therefore, under stress-induced conditions in the middle ear, this NTHi lipoprotein may be expressed.

TABLE 3A Genbank Contig E score Hit Identity Organism Protein SEQ ID NO: 104 4.00E−59 CpdB Pasteurella NP_246953.1 nt. 204-659 of multocida SEQ ID NO: 104 106 9.00E−10 hypothetical protein Pyrococcus G71244 nt. 40-309 of PH0217 - horikoshii SEQ ID NO: 106 106 1.00E−08 unknown Pasteurella NP_246871.1 nt. 605-694 of multocida SEQ ID NO: 106 106 2.00E−20 Orf122 Chlorobium AAG12204.1 nt. 7-210 of tepidum SEQ ID NO: 106 110 3.00E−05 ArdC antirestriction IncW plasmid pSa AAD52160.1 compliment of protein nt. 959-1162 of SEQ ID NO: 110 110 1.00E−33 hypothetical protein Salmonella NP_458676.1 compliment of enterica subsp. nt. nt. 181-825 enterica serovar of SEQ ID NO: Typhi 110 111 5.00E−12 putative membrane Salmonella NP_458664.1 compliment of protein enterica subsp. nt. 45-287 of enterica serovar SEQ ID NO: 111 Typhi 111 6.00E−41 hypothetical protein Salmonella NP_458658.1 compliment of enterica subsp. nt. 1091-1480 of enterica serovar SEQ ID NO: 111 Typhi 114 7.00E−80 unknown Pasteurella NP_245828.1 compliment of multocida nt. 118-696 of SEQ ID NO: 114 115 2.00E−09 A111R Paramecium NP_048459.1 nt. 555-869 of bursaria Chlorella SEQ ID NO: 115 virus 1 118 5.00E−45 DNA methylase Vibrio cholerae NP_231404.1 nt. 44-439 of HsdM, putative SEQ ID NO: 118 122 2.00E−18 unknown Pasteurella NP_245314.1 nt. 865-1302 of multocida SEQ ID NO: 122 123 4.00E−99 RNA Proteus mirabilis P50509 nt. 351-782 of POLYMERASE SEQ ID NO: 123 SIGMA-32 FACTOR 124 9.00E−58 ACETOLACTATE Spirulina platensis P27868 nt. 603-1025 of SYNTHASE SEQ ID NO: 124 (ACETOHYDROXY- ACID SYNTHASE) (ALS) 130 0 restriction Neisseria CAA09003.1 nt. 495-1559 of modification meningitidis SEQ ID NO: 130 system-R protein 131 6.00E−91 uronate isomerase Salmonella NP_457532.1 compliment of (glucuronate enterica subsp. nt. 661-1380 of isomerase) enterica serovar SEQ ID NO: 131 Typhi 133 3.00E−30 GyrA Pasteurella NP_245778.1 compliment of multocida nt. 1447-1626 of SEQ ID NO: 133 133 1.00E−27 DNA GYRASE Pectobacterium P41513 compliment of SUBUNIT A carotovorum nt. 1302-1442 of SEQ ID NO: 133 138 7.00E−06 KicA Pasteurella NP_245545.1 compliment of multocida nt. 92-157 of SEQ ID NO: 138 138 1.00E−148 TYPE II Haemophilus O30869 compliment of RESTRICTION aegyptius nt. 164-1045 of ENZYME HAEII SEQ ID NO: 138 (ENDONUCLEASE HAEII) (R. HAEII) 143 4.00E−06 Gifsy-1 prophage Salmonella NP_461555.1 compliment of protein typhimurium LT2 nt. 228-632 of SEQ ID NO: 143 143 1.00E−14 hypothetical protein Bacteriophage NP_050531.1 compliment of VT2-Sa nt. 778-1248 of SEQ ID NO: 143 143 5.00E−09 hypothetical protein Salmonella CAD09979.1 compliment of enterica subsp. nt. 715-1026 of enterica serovar SEQ ID NO: 143 Typhi 143 6.00E−10 hypothetical 14.9 kd Escherichia coli NP_065324.1 nt. 3-173 of protein SEQ ID NO: 143 147 1.00E−38 GTP-binding Escherichia coli NP_289127.1 compliment of elongation factor, O157:H7 EDL933 nt. 172-342 of may be inner SEQ ID NO: 147 membrane protein 147 2.00E−14 GTP-binding Borrelia NP_212222.1 compliment of membrane protein burgdorferi nt. 17-181 of (lepA) SEQ ID NO: 147 148 6.00E−17 galactokinase Homo sapiens AAC35849.1 compliment of nt. 746-1246 of SEQ ID NO: 148 148 7.00E−96 GALACTOKINASE Actinobacillus P94169 compliment of (GALACTOSE pleuropneumoniae nt. 232-741 of KINASE) SEQ ID NO: 148 149 1.00E−92 GTP-binding Buchnera sp. NP_240245.1 compliment of protein TypA/BipA APS nt. 265-1077 of SEQ ID NO: 149 15 2.00E−21 ORF1 Escherichia coli CAA39631.1 nt. 665-850 of SEQ ID NO: 15 150 6.00E−17 unknown Pasteurella NP_245919.1 nt. 171-665 of multocida SEQ ID NO: 150 153 7.00E−07 cuter membrane Rickettsia conorii T30852 nt. 51-623 of protein A SEQ ID NO: 153 155 7.00E−40 cytochrome d Vibrio cholerae NP_233259.1 nt. 583-1002 of ubiquinol oxidase, SEQ ID NO: 155 subunit II 157 7.00E−13 unknown Pasteurella NP_245490.1 compliment of multocida nt. 1170-1367 of SEQ ID NO: 157 157 2.00E−05 glycosyl Neisseria AAA68012.1 nt. 85-189 of transferase gonorrhoeae SEQ ID NO: 157 158 1.00E−152 MltC Pasteurella NP_246259.1 compliment of multocida nt. 36-530 of SEQ ID NO: 158 161 3.00E−25 lipoprotein, putative Vibrio cholerae NP_230232.1 nt. 870-1439 of SEQ ID NO: 161 163 9.00E−53 chorismate Caulobacter NP_421948.1 nt. 1283-2029 of synthase crescentus SEQ ID NO: 163 168 3.00E−13 COPPER- Mus musculus Q64430 nt. 66-995 of TRANSPORTING SEQ ID NO: 168 ATPASE 1 (COPPER PUMP 1) 168 2.00E−22 Cu transporting Homo sapiens 2001422A nt. 135-989 of ATPase P SEQ ID NO: 168 174 8.00E−48 magnesium/cobalt Mesorhizobium NP_103977.1 nt. 918-1205 of transport protein loti SEQ ID NO: 174 175 5.00E−26 vacB protein Buchnera sp. NP_240369.1 compliment of APS nt. 1-1587 of SEQ ID NO: 175 176 3.00E−21 putative ABC Campylobacter NP_282774.1 compliment of transport system jejuni nt. 259-1089 of permease protein [ SEQ ID NO: 176 183 5.00E−29 PROBABLE ATP Ralstonia NP_521442.1 compliment of SYNTHASE A solanacearum nt. 42-677 of CHAIN SEQ ID NO: 183 TRANSMEMBRANE PROTEIN 185 6.00E−85 putative exported Salmonella NP_458655.1 compliment of protein enterica subsp. nt. 162-1529 of enterica serovar SEQ ID NO: 185 Typhi 187 2.00E−05 transketolase Homo sapiens AAA61222.1 nt. 709-819 of SEQ ID NO: 187 188 1.00E−116 ribonuclease E Xylella fastidiosa NP_299884.1 compliment of 9a5c nt. 280-1704 of SEQ ID NO: 188 192 1.00E−38 ImpA Pasteurella NP_245829.1 nt. 35-448 of multocida SEQ ID NO: 192 193 3.00E−08 Orf80 Enterobacteria NP_052285.1 nt. 1612-1818 of phage 186 SEQ ID NO: 193 193 1.00E−06 holin Haemophilus AAC45168.1 nt. 370-576 of somnus SEQ ID NO: 193 193 0.007 unknown Enterobacteria NP_052260.1 nt. 1376-1609 of phage 186 SEQ ID NO: 193 193 2.00E−48 lysozyme Haemophilus AAC45169.1 nt. 608-1093 of somnus SEQ ID NO: 193 199 4.00E−21 unknown protein Escherichia coli NP_288675.1 nt. 398-778 of O157:H7 SEQ ID NO: 199 EDL933, prophage CP- 933V 199 2.00E−49 hypothetical protein Bacteriophage NP_049495.1 compliment of 933W nt. 1907-2392 of SEQ ID NO: 199 20 1.00E−62 RpL14 Pasteurella NP_246344.1 compliment of multocida nt. 233-601 of SEQ ID NO: 20 200 2.00E−62 hypothetical protein Salmonella NP_458658.1 compliment of enterica subsp. nt. 431-997 of enterica serovar SEQ ID NO: 200 Typhi 200 3.00E−16 hypothetical protein Salmonella NP_458657.1 compliment of enterica subsp. nt. 1028-1264 of enterica serovar SEQ ID NO: 200 Typhi 201 2.00E−26 TsaA Pasteurella NP_245732.1 compliment of multocida nt. 1618-1809 of SEQ ID NO: 201 209 6.00E−16 TsaA Pasteurella NP_245732.1 compliment of multocida nt. 2-136 of SEQ ID NO: 209 211 2.00E−15 unknown Pasteurella NP_245535.1 compliment of multocida nt. 23-211 of SEQ ID NO: 211 211 1.00E−70 PUTATIVE Ralstonia NP_520082.1 compliment of ATPASE PROTEIN solanacearum nt. 475-915 of SEQ ID NO: 211 212 3.00E−18 hypothetical protein Escherichia coli NP_309775.1 compliment of O157:H7 nt. 895-1035 of SEQ ID NO: 212 216 1.00E−173 unknown Pasteurella NP_245069.1 nt. 35-1543 of multocida SEQ ID NO: 216 217 9.00E−18 diacylglycerol Vibrio cholerae NP_233101.1 nt. 2083-2208 of kinase SEQ ID NO: 217 221 4.00E−34 Tail-Specific Chlamydia NP_219953.1 nt. 849-1421 of Protease trachomatis SEQ ID NO: 221 222 4.00E−23 AGR_C_3689p Agrobacterium NP_355005.1 compliment of tumefaciens str. nt. 940-1305 of C58 (Cereon) SEQ ID NO: 222 224 9.00E−19 unknown Pasteurella NP_245536.1 nt. 15-308 of multocida SEQ ID NO: 224 225 1.00E−89 portal vector-like Salmonella NP_461651.1 nt. 31-750 of of protein, in phage typhimurium SEQ ID NO: 225 P2 [Salmonella LT2Fels-2 typhimurium LT2] prophage 229 2.00E−25 anaerobic Salmonella CAB62266.1 nt. 1806-2108 of ribonucleotide typhimurium SEQ ID NO: 229 reductase 234 3.00E−08 conserved Xylella fastidiosa NP_299850.1 nt. 1680-2048 of hypothetical protein 9a5c SEQ ID NO: 234 234 1.00E−42 Methionine Clostridium NP_348177.1 compliment of sulfoxide reductase acetobutylicum nt. 415-654 of C-terminal domain SEQ ID NO: 234 related protein, YPPQ ortholog 235 4.00E−16 phage-related tail Wolbachia AAK85310.1 compliment of protein endosymbiont of nt. 931-1929 of Drosophila SEQ ID NO: 235 melanogaster 235 6.00E−56 similar to orfG Salmonella NP_461625.1 compliment of protein in phage typhimurium LT2, nt. 313-1863 of 186, Salmonella Fels-2 prophage SEQ ID NO: 235 typhimurium LT2 236 6.00E−20 conserved Pseudomonas NP_252693.1 nt. 1572-1916 hypothetical protein aeruginosa of SEQ ID NO: 236 240 5.00E−27 MODIFICATION Brevibacterium P10283 compliment of METHYLASE BEPI epidermidis nt. 922-1305 of SEQ ID NO: 240 241 2.00E−15 phage-related Xylella fastidiosa NP_299573.1 compliment of protein 9a5c nt. 865-1305 of SEQ ID NO: 241 241 4.00E−08 hypothetical protein phage SPP1 T42296 nt. 73-636 of SEQ ID NO: 241 241 4.00E−07 hypothetical protein Salmonella NP_458686.1 nt. 10-468 of enterica subsp. SEQ ID NO: 241 enterica serovar Typhi 242 2.00E−29 translation chloroplast - S35701 compliment of elongation factor soybean nt. 18-1085 of EF-G SEQ ID NO: 242 247 3.00E−23 GTP Synechococcus Q54769 compliment of CYCLOHYDROLASE sp. PCC 7942 nt. 1009-1257c I (GTP-CH-I) of SEQ ID NO: 247 248 6.00E−05 phospho-N- Aquifex aeolicus NP_213025.1 nt. 830-1747 of acetylmuramoyl- SEQ ID NO: 248 pentapeptide- transferase 25 2.00E−86 PROBABLE Ralstonia NP_522358.1 compliment of TRANSPORT solanacearum nt. 309-854 of TRANSMEMBRANE SEQ ID NO: 25 PROTEIN 25 7.00E−06 major facilitator Caulobacter NP_419155.1 compliment of family transporter crescentus nt. 134-283 of SEQ ID NO: 25 250 1.00E−150 CpdB Pasteurella NP_246953.1 compliment of multocida nt. 36-1016 of SEQ ID NO: 250 252 3.00E−57 alanyl-tRNA Vibrio cholerae AAA99922.1 compliment of synthetase nt. 1418-1951 of SEQ ID NO: 252 253 1.00E−108 similar to Listeria NP_464432.1 compliment of glutathione monocytogenes nt. 411-1358 of Reductase EGD-e of SEQ ID NO: 253 259 3.00E−39 hypothetical protein Salmonella NP_458654.1 compliment of enterica subsp. nt. 342-1037 of enterica serovar SEQ ID NO: 259 Typhi 259 3.00E−17 possible exported Salmonella NP_458653.1 compliment of protein enterica subsp. nt. 1251-1607 enterica serovar of SEQ ID NO: Typhi 259 261 5.00E−74 hypothetical protein Haemophilus S27582 compliment of 6 - Haemophilus influenzae nt. 3-422 of influenzae SEQ ID NO: 261 263 1.00E−94 putative Haemophilus AAD01406.1 nt. 2142-2672 of transposase paragallinarum SEQ ID NO: 263 264 1.00E−126 unknown Actinobacillus NP_067554.1 nt. 40-714 of actinomycetemcomitans SEQ ID NO: 264 264 1.00E−103 unknown Actinobacillus NP_067555.1 nt. 695-1309 of actinomycetemcomitans SEQ ID NO: 264 264 2.00E−21 unknown Actinobacillus NP_067556.1 nt. 1302-1448 of actinomycetemcomitans SEQ ID NO: 264 265 6.00E−27 Aminopeptidase 2 chloroplast Q42876 nt. 556-1539 of SEQ ID NO: 265 268 1.00E−116 MutY Pasteurella NP_246257.1 nt. 1003-1581 of multocida SEQ ID NO: 268 272 1.00E−07 hypothetical protein Bacteriophage NP_049495.1 compliment of 933W nt. 77-232 of SEQ ID NO: 272 274 3.00E−13 unknown Pasteurella NP_246952.1 compliment of multocida nt. 1658-1975 of SEQ ID NO: 274 275 3.00E−20 CafA Neisseria AAG24267.1 nt. 1299-1571 of gonorrhoeae SEQ ID NO: 275 276 1.00E−45 mukE protein Vibrio cholerae NP_231351.1 compliment of nt. 650-1390 of SEQ ID NO: 276 276 1.00E−69 KicA Pasteurella NP_245545.1 compliment of multocida nt. 647-1321 of SEQ ID NO: 276 278 2.00E−56 3-oxoacyl-[acyl- Salmonella NP_455686.1 nt. 1366-1944 of carrier-protein] enterica subsp. SEQ ID NO: 278 synthase III enterica serovar Typhi 281 5.00E−56 unknown Pasteurella NP_246261.1 compliment of multocida nt. 31-678 of SEQ ID NO: 281 282 3.00E−09 orf25; similar to T bacteriophage phi NP_490625.1 compliment of gene of P2 CTX nt. 511-1032 of SEQ ID NO: 282 282 1.00E−08 orf11; similar to Haemophilus AAC45165.1 compliment of phage P2 gene S- somnus nt. 1450-1584 of like product, which SEQ ID NO: 282 is involved in tail synthesis, 282 9.00E−27 putative Salmonella NP_457167.1 compliment of bacteriophage tail enterica subsp. nt. 3-509 of protein enterica serovar SEQ ID NO: 282 Typhi 286 5.00E−18 plasmid-related Listeria innocua NP_471066.1 compliment of protein plasmid nt. 887-1501 of SEQ ID NO: 286 287 8.00E−20 GTP Escherichia coli NP_287920.1 nt. 2-145 of cyclohydrolase II O157:H7 EDL933 SEQ ID NO: 287 289 1.00E−168 MODIFICATION Haemophilus O30868 compliment of METHYLASE aegyptius nt. 138-1091 of HAEII SEQ ID NO: 289 289 5.00E−11 TYPE II Haemophilus O30869 compliment of RESTRICTION aegyptius nt. 22-132 of ENZYME HAEII SEQ ID NO: 289 289 6.00E−47 mukF homolog Haemophilus AAB70828.1 compliment of influenzae biotype nt. 1107-1385 aegyptius of SEQ ID NO: 289 294 1.00E−171 LICA PROTEIN Haemophilus P14181 compliment of influenzae nt. 677-1564 of RM7004 SEQ ID NO: 294 297 1.00E−158 DNA methylase Vibrio cholerae NP_231404.1 compliment of HsdM, putative nt. 12-1136 of SEQ ID NO: 297 302 0 HEME-BINDING Haemophilus P33950 nt.3-1316 of PROTEIN A influenzae DL42 SEQ ID NO: 302 304 6.00E−19 hypothetical protein 6 Haemophilus S27582 nt. 121-267 of influenzae SEQ ID NO: 304 305 6.00E−40 putative Streptococcus NP_269557.1 nt. 65-805 of recombinase - pyogenes M1 SEQ ID NO: 305 phage associated GAS 305 7.00E−22 single stranded Shewanella sp. AAB57886.1 nt. 1607-2014 of DNA-binding F1A SEQ ID NO: 305 protein 305 1.00E−43 phage-related Bacillus NP_244410.1 nt. 92-751 of protein halodurans SEQ ID NO: 305 312 1.00E−28 PUTATIVE Ralstonia NP_518994.1 nt. 1819-2673 of BACTERIOPHAGE- solanacearum SEQ ID NO: 312 RELATED TRANSMEMBRANE PROTEIN 312 9.00E−25 similar to Homo sapiens XP_068727.1 nt. 27-1001 of BASEMENT SEQ ID NO: 312 MEMBRANE- SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR (HSPG) 315 2.00E−45 uracil permease Deinococcus NP_296001.1 compliment of radiodurans nt. 525-1592 of SEQ ID NO: 315 318 7.00E−15 CzcD Pasteurella NP_246276.1 compliment of multocida nt. 3-227 of SEQ ID NO: 318 320 2.00E−60 orf3; similar to Haemophilus AAC45159.1 compliment of endonuclease somnus nt. 606-1241 of subunit of the SEQ ID NO: 320 phage P2 terminase (gene M) 320 2.00E−09 orf4; similar to Haemophilus AAC45160.1 compliment of head somnus nt. 52-285 of completion/stabilization SEQ ID NO: 320 protein (gene L) of phage P2 320 3.00E−35 orf2; similar to Haemophilus AAC45158.1 compliment of major capsid somnus nt. 1271-1624 of protein precursor of SEQ ID NO: 320 phage P2 (gene N) 323 4.00E−37 dedC protein Escherichia coli AAA23966.1 compliment of nt. 74-463 of SEQ ID NO: 323 324 1.00E−153 conserved Neisseria NP_274972.1 compliment of hypothetical protein meningitidis nt. 930-1943 of MC58 SEQ ID NO: 324 326 5.00E−52 selenophosphate Eubacterium CAB53511.1 compliment of synthetase acidaminophilum nt. 1186-2292 of SEQ ID NO: 326 328 1.00E−129 secretion protein Pseudomonas NP_252510.1 compliment of SecD aeruginosa nt. 8-625 of SEQ ID NO: 328 333 3.00E−08 unknown Pasteurella NP_245489.1 compliment of multocida nt. 5-418 of SEQ ID NO: 333 336 6.00E−38 probable methyl Pseudomonas NP_253353.1 compliment of transferase aeruginosa nt. 2547-2819 of SEQ ID NO: 336 338 2.00E−98 Pmi Pasteurella NP_245766.1 nt. 144-842 of multocida SEQ ID NO: 338 339 2.00E−07 tRNA Escherichia coli QQECPE nt. 2331-2540 of nucleotidyltransferase SEQ ID NO: 339 340 0 DNA gyrase, Salmonella NP_461214.1 compliment of subunit A, type II typhimurium LT2 nt. 93-1799 of topoisomerase SEQ ID NO: 340 342 4.00E−12 tolA protein Haemophilus JC5212 nt. 980-1318 of influenzae SEQ ID NO: 342 344 1.00E−07 MODIFICATION Haemophilus P50192 compliment of METHYLASE parahaemolyticus nt. 849-1034 of HPHIA SEQ ID NO: 344 344 8.00E−05 ABC transporter Leishmania major AAF31030.1 compliment of protein 1 nt. 17-205 of SEQ ID NO: 344 349 3.00E−44 conserved Neisseria NP_273467.1 compliment of hypothetical protein meningitidis nt. 1397-1903 of MC58 SEQ ID NO: 349 349 8.00E−09 hypothetical protein Pseudomonas NP_252667.1 compliment of aeruginosa nt. 795-1121 of SEQ ID NO: 349 349 9.00E−10 conserved Helicobacter NP_207009.1 compliment of hypothetical pylori 26695 nt. 1319-1816 of secreted protein SEQ ID NO: 349 349 2.00E−06 putative TPR Salmonella NP_463149.1 compliment of repeat protein typhimurium LT2 nt. 2244-2558 of SEQ ID NO: 349 35 1.00E−23 type I restriction- Xylella fastidiosa NP_300003.1 compliment of modification 9a5c nt. 29-388 of system specificity SEQ ID NO: 35 determinant 352 1.00E−116 putative peptidase Escherichia coli NP_416827.1 compliment of K12 nt. 951-1640 of SEQ ID NO: 352 352 0 unknown Pasteurella NP_245275.1 compliment of multocida nt. 86-946 of SEQ ID NO: 352 354 5.00E−86 putative uronate Salmonella NP_462052.1 compliment of isomerase typhimurium LT2 nt. 168-914 of SEQ ID NO: 354 356 1.00E−07 isomerase-like Escherichia coli S57220 nt. 5-73 of protein (DsbD) - SEQ ID NO: 356 358 1.00E−07 USG protein Pediococcus CAC16793.1 nt.534-1307 of pentosaceus SEQ ID NO: 358 358 0.005 HsdS protein Escherichia coli CAA10700.1 nt. 26-205 of SEQ ID NO: 358 361 1.00E−152 maltodextrin Escherichia coli NP_289957.1 compliment of phosphorylase O157:H7 EDL933 nt. 77-922 of SEQ ID NO: 361 363 6.00E−06 BH2505-unknown Bacillus NP_243371.1 nt. 554-844 of conserved protein halodurans SEQ ID NO: 363 368 1.00E−12 H02F09.3.p Caenorhabditis NP_508295.1 compliment of elegans nt. 1069-1977 of SEQ ID NO: 368 368 6.00E−27 hypothetical Mesorhizobium NP_102360.1 compliment of glycine-rich protein loti nt. 1201-1986 of SEQ ID NO: 368 37 6.00E−09 putative ATP- Escherichia coli NP_415469.1 compliment of binding component K12 nt. 455-691 of of a transport SEQ ID NO: 37 system 372 7.00E−18 conserved Clostridium BAB80319.1 compliment of hypothetical protein perfringens nt. 1763-1924 of SEQ ID NO: 372 376 7.00E−24 putative Salmonella NP_456379.1 compliment of bacteriophage enterica subsp. nt. 158-808 of protein enterica serovar SEQ ID NO: 376 Typhi 376 8.00E−10 hypothetical protein Xylella fastidiosa NP_298882.1 compliment of 9a5c nt. 1129-1671 of SEQ ID NO: 376 376 9.00E−06 lin1713 Listeria innocua NP_471049.1 compliment of nt 913-1557 of SEQ ID NO: 376 377 6.00E−05 Vng1732c Halobacterium sp. NP_280487.1 nt. 2378-2587 of NRC-1 SEQ ID NO: 377 377 1.00E−11 INVASIN Yersinia P31489 compliment of PRECURSOR enterocolitica nt. 127-345 of (OUTER SEQ ID NO: 377 MEMBRANE ADHESIN) 382 4.00E−16 unknown Pasteurella NP_246871.1 compliment of multocida nt. 967-1068 of SEQ ID NO: 382 383 4.00E−36 putative Streptomyces BAB69302.1 nt. 488-1162 of transposase avermitilis SEQ ID NO: 383 383 3.00E−58 recombinase IncN plasmid R46 NP_511241.1 compliment of nt. 1-393 of SEQ ID NO: 383 383 4.00E−24 transposase Escherichia coli I69674 nt. 1294-1740 of SEQ ID NO: 383 383 0 tnpA Yersinia CAA73750.1 nt. 1782-2834 of enterocolitica SEQ ID NO: 383 385 2.00E−31 unknown Pasteurella NP_246065.1 nt. 1515-1772 of multocida SEQ ID NO: 385 386 5.00E−65 cydC [ Escherichia coli AAA66172.1 compliment of nt. 3438-4115 of SEQ ID NO: 386 386 4.00E−33 ABC transporter, Mesorhizobium NP_105463.1 compliment of ATP-binding loti nt. 2569-3390 of protein SEQ ID NO: 386 388 3.00E−45 60 KDA INNER- Coxiella burnetii P45650 compliment of MEMBRANE nt. 3211-3759 PROTEIN of SEQ ID NO: HOMOLOG 388 390 4.00E−25 putative DNA- Salmonella NP_458175.1 nt. 1051-1416 of binding protein enterica subsp. SEQ ID NO: 390 enterica serovar Typhi 390 3.00E−13 transcriptional Bacillus NP_241773.1 compliment of regulator halodurans nt. 84-578 of SEQ ID NO: 390 390 3.00E−06 DNA translocase Staphylococcus NP_372265.1 compliment of stage III sporulation aureus subsp. nt. 620-871 of prot homolog aureus Mu50 SEQ ID NO: 390 395 7.00E−31 ATPase, Cu++ Homo sapiens NP_000044.1 compliment of transporting, beta nt. 615-1406 of polypeptide SEQ ID NO: 395 397 3.00E−23 terminase large Bacteriophage NP_112076.1 compliment of subunit HK620 nt. 2363-2725 of SEQ ID NO: 397 397 3.00E−16 hypothetical protein Xylella fastidiosa NP_297824.1 compliment of 9a5c nt. 1517-1744 of SEQ ID NO: 397 398 4.00E−67 orf32 Haemophilus NP_536839.1 compliment of phage HP2 nt. 1288-1866 of SEQ ID NO: 398 398 8.00E−24 putative Salmonella NP_463063.1 compliment of cytoplasmic protein typhimurium LT2 nt. 798-1220 of SEQ ID NO: 398 398 2.00E−83 orf31 Haemophilus NP_043502.1 compliment of phage HP1 nt. 1881-2510 of SEQ ID NO: 398 399 1.00E−94 HEME/HEMOPEXIN- Haemophilus P45355 nt. 88-774 of BINDING influenzae N182 SEQ ID NO: 399 PROTEIN 401 3.00E−63 Sty SBLI Salmonella CAA68058.1 nt. 1690-2742 of enterica SEQ ID NO: 401 401 3.00E−06 RESTRICTION- Mycoplasma NP_325912.1 nt. 79-489 of MODIFICATION pulmonis SEQ ID NO: 401 ENZYME SUBUNIT M3 402 2.00E−13 OPACITY Neisseria Q05033 compliment of PROTEIN OPA66 gonorrhoeae nt. 2634-2915 of PRECURSOR SEQ ID NO: 402 406 8.00E−13 type I restriction Neisseria NP_273876.1 nt. 281-520 of enzyme EcoR124IIR meningitidis SEQ ID NO: 406 MC58 407 6.00E−65 unknown Pasteurella NP_246237.1 nt. 938-2450 of multocida SEQ ID NO: 407 407 5.00E−99 PepE Pasteurella NP_245391.1 nt. 1216-1917 of multocida SEQ ID NO: 407 407 1.00E−16 Hemoglobin- Haemophilus Q48153 nt. 1-141 of haptoglobin binding influenzae Tn106 SEQ ID NO: 407 protein A 409 1.00E−106 hypothetical protein 1 Haemophilus S27577 compliment of influenzae nt. 2524-3159 of SEQ ID NO: 409 411 4.00E−29 heme-repressible Haemophilus AAB46794.1 nt. 391-615 of hemoglobin-binding influenzae, type b, SEQ ID NO: 411 protein strain HI689 411 0 Hemoglobin- Haemophilus Q48153 nt. 651-3263 of haptoglobin binding influenzae Tn106 SEQ ID NO: 411 protein A 412 2.00E−07 REGULATORY bacteriophage P03036 compliment of PROTEIN CRO 434 nt. 59-259 of (ANTIREPRESSOR) SEQ ID NO: 412 412 4.00E−06 hypothetical protein Bacteriophage CAC83535.1 nt. 1436-1654 of P27 SEQ ID NO: 412 413 8.00E−07 hypothetical protein Deinococcus NP_294301.1 compliment of radiodurans nt. 791-1012 of SEQ ID NO: 413 414 9.00E−65 conserved Vibrio cholerae NP_230092.1 nt. 1696-2103 of hypothetical protein SEQ ID NO: 414 414 3.00E−93 unknown Pasteurella NP_246834.1 nt. 1777-2109 of multocida SEQ ID NO: 414 416 2.00E−17 unknown Pasteurella NP_246629.1 compliment of multocida nt. 2565-2831 of SEQ ID NO: 416 416 4.00E−26 hypothetical protein Escherichia coli S30728 compliment of o154 nt. 1928-2254 of SEQ ID NO: 416 416 3.00E−37 transport protein Pseudomonas NP_253757.1 compliment of TatC aeruginosa nt. 1494-2018 of of SEQ ID NO: 416 417 1.00E−66 weakly similar to Listeria innocua NP_471073.1 compliment of methyltransferases nt. 999-1928 of SEQ ID NO: 417 417 5.00E−05 DNA-BINDING Pectobacterium Q47587 compliment of PROTEIN RDGA carotovorum nt. 3526-4212 of SEQ ID NO: 417 417 2.00E−29 putative phage- Yersinia pestis NP_407132.1 compliment of related protein nt. 2546-2938 of SEQ ID NO: 417 417 3.00E−06 Adenine-specific Thermoplasma NP_393798.1 compliment of DNA methylase acidophilum nt. 826-1020 of SEQ ID NO: 417 43 9.00E−16 PcnB Pasteurella NP_245801.1 nt. 511-870 of multocida SEQ ID NO: 43 434 2.00E−97 beta′ subunit of Nephroselmis NP_050840.1 compliment of RNA polymerase olivacea nt. 32-1534 of SEQ ID NO: 434 435 4.00E−52 MODIFICATION Brevibacterium P10283 compliment of METHYLASE BEPI epidermidis nt. 11-565 of SEQ ID NO: 435 435 4.00E−57 pentafunctional Saccharomyces NP_010412.1 compliment of arom polypeptide cerevisiae nt. 757-2064 of (contains: 3- SEQ ID NO: 435 dehydroquinate synthase, 3- dehydroquinate dehydratase (3- dehydroquinase), shikimate 5- dehydrogenase, shikimate kinase, and epsp synthase) 437 5.00E−70 dihydrofolate Haemophilus S52336 nt. 2393-2767 of reductase influenzae SEQ ID NO: 437 (clinical isolate R1042) 438 1.00E−106 polyA polymerase Vibrio cholerae NP_230244.1 nt. 3-1124 of SEQ ID NO: 438 439 6.00E−60 Porphyrin Salmonella NP_457816.1 nt. 2343-2783 of biosynthetic protein enterica subsp. SEQ ID NO: 439 enterica serovar Typhi 441 5.00E−73 RimM Pasteurella NP_246234.1 compliment of multocida nt. 151-441 of SEQ ID NO: 441 442 9.00E−31 LIPOPROTEIN Salmonella P40827 compliment of NLPD typhimurium nt. 3362-3520 of SEQ ID NO: 442 444 6.00E−24 glycine betaine Staphylococcus NP_371872.1 compliment of transporter aureus subsp. nt. 2242-2514 of aureus Mu50 SEQ ID NO: 444 452 6.00E−28 unknown Pasteurella NP_245616.1 compliment of multocida nt. 533-883 of SEQ ID NO: 452 452 0 Type I restriction Escherichia coli Q47163 nt. 3291-4154 of enzyme Ecoprrl M SEQ ID NO: 452 protein 452 2.00E−75 type I restriction Ureaplasma NP_077929.1 nt. 4156-4662 of enzyme M protein urealyticum SEQ ID NO: 452 455 9.00E−56 PROBABLE Ralstonia NP_520059.1 nt. 2028-2774 of BACTERIOPHAGE solanacearum SEQ ID NO: 455 PROTEIN 455 2.00E−55 orf2; similar to Haemophilus AAC45158.1 nt. 2864-3490 of major capsid somnus SEQ ID NO: 455 protein precursor of phage P2 (gene N), 455 1.00E−175 gpP Enterobacteria NP_046758.1 compliment of phage P2 nt. 127-1812 of SEQ ID NO: 455 456 1.00E−38 hypothetical protein Pseudomonas NP_542872.1 compliment of putida nt. 1010-1282 of SEQ ID NO: 456 456 1.00E−172 hypothetical protein Pseudomonas NP_542873.1 compliment of putida nt. 1443-2006 of SEQ ID NO: 546 457 1.00E−116 hypothetical protein Haemophilus S15287 compliment of (galE 5′ region) - influenzae nt. 62-961 of Haemophilus SEQ ID NO: 457 influenzae 457 1.00E−134 dTDPglucose 4,6- Actinobacillus T00102 nt. 2637-3656 of dehydratase actinomycetemcomitans SEQ ID NO: 457 459 2.00E−10 RNA polymerase Synechocystis sp. NP_441586.1 nt. 25-117 of gamma-subunit PCC 6803 SEQ ID NO: 459 461 9.00E−51 conserved Staphylococcus NP_370593.1 nt. 4124-4624 of hypothetical protein aureus subsp. SEQ ID NO: 461 aureus Mu50 462 9.00E−06 NADH Burkholderia AAG01016.1 nt. 703-828 of dehydrogenase pseudomallei SEQ ID NO: 462 465 3.00E−41 GTP-binding Synechocystis sp. NP_441951.1 compliment of protein Era PCC 6803 nt. 2470-2787 of SEQ ID NO: 465 466 1.00E−15 putative Salmonella NP_455548.1 nt. 837-1478 of bacteriophage enterica subsp. SEQ ID NO: 466 protein enterica serovar Typhi 466 1.00E−90 orf31 Haemophilus NP_043502.1 nt. 2396-3199 of phage HP1 SEQ ID NO: 466 469 0 Hemoglobin and Haemophilus Q9X442 compliment of hemoglobin- influenzae HI689 nt. 427-3459 of haptoglobin binding SEQ ID NO: 469 protein C precursor 471 8.00E−05 transposase, Neisseria NP_274608.1 nt. 2957-3217 of putative meningitidis SEQ ID NO: 471 MC58 472 6.00E−08 hypothetical protein Salmonella NP_458660.1 compliment of enterica subsp. nt. 2881-3270 of enterica serovar SEQ ID NO: 472 Typhi 472 5.00E−23 antirestriction Mesorhizobium NP_106707.1 nt. 4908-5324 of protein loti SEQ ID NO: 472 472 1.00E−75 hypothetical protein Salmonella NP_458661.1 compliment of enterica subsp. nt. 1931-2776 of enterica serovar SEQ ID NO: 472 Typhi 472 9.00E−72 hypothetical protein Salmonella NP_458662.1 compliment of enterica subsp. nt. 544-1689 of enterica serovar SEQ ID NO: 472 Typhi 475 3.00E−25 unknown Pasteurella NP_244952.1 nt. 3207-3626 of multocida SEQ ID NO: 475 476 8.00E−73 putative DNA- Salmonella NP_458175.1 compliment of binding protein enterica subsp. nt. 3339-4310 of enterica serovar SEQ ID NO: 476 Typhi 476 6.00E−47 anticodon nuclease Neisseria NP_273873.1| compliment of meningitidis nt. 4397-4885 of MC58 SEQ ID NO: 476 478 3.00E−06 methionin Arabidopsis CAB38313.1 compliment of synthase-like thaliana nt. 3554-3679 of enzyme SEQ ID NO: 478 478 3.00E−05 unknown Pasteurella NP_245444.1 compliment of multocida nt. 164-250 of SEQ ID NO: 478 479 1.00E−18 conserved Xylella fastidiosa NP_298841.1 nt. 2302-2658 of hypothetical protein 9a5c SEQ ID NO: 479 48 3.00E−19 Dca Neisseria AAF12796.1 compliment of gonorrhoeae nt. 225-746 of SEQ ID NO: 48 482 1.00E−06 hypothetical protein Neisseria NP_275122.1 nt. 2055-2189 of meningitidis SEQ ID NO: 482 MC58 482 9.00E−28 conserved Neisseria NP_274383.1 nt. 1689-1898 of hypothetical protein meningitidis SEQ ID NO: 482 MC58 487 5.00E−75 conserved Neisseria NP_284304.1 nt. 2541-2978 of hypothetical protein meningitidis SEQ ID NO: 487 Z2491 488 2.00E−64 unknown Pasteurella NP_246617.1 nt. 2983-3540 of multocida SEQ ID NO: 488 488 8.00E−93 1-deoxy-D-xylulose Zymomonas AAD29659.1 nt. 1344-1880 of 5-phosphate mobilis SEQ ID NO: 488 reductoisomerase 491 5.00E−51 rubredoxin Clostridium AAB50346.1 compliment of oxidoreductase acetobutylicum nt. 1690-2439 of homolog SEQ ID NO: 491 492 1.00E−27 phosphotransferase Staphylococcus AAK83253.1 compliment of system enzyme aureus nt. 755-970 of IIA-like protein SEQ ID NO: 492 493 2.00E−84 unknown Actinobacillus AAC70895.1 nt. 3333-3935 of actinomycetemcomitans SEQ ID NO: 493 493 4.00E−49 unknown Helicobacter NP_223898.1 nt. 3345-4010 of pylori J99 SEQ ID NO: 493 493 9.00E−31 transcriptional Acinetobacter AAF20290.1 nt. 1885-2793 of factor MdcH calcoaceticus SEQ ID NO: 493 493 6.00E−30 HimA Pasteurella NP_245565.1 nt. 1129-1260 of multocida SEQ ID NO: 493 494 4.00E−85 putative prophage Yersinia pestis NP_404712.1 nt. 900-2099 of integrase SEQ ID NO: 494 494 4.00E−63 DNA Xylella fastidiosa NP_299063.1 compliment of methyltransferase 9a5c nt. 5544-6170 of SEQ ID NO: 494 494 6.00E−19 MODIFICATION Lactococcus lactis P34877 compliment of METHYLASE subsp. cremoris nt. 5019-6113 of SCRFIA SEQ ID NO: 494 497 0 transferrin-binding Haemophilus S70906 nt. 3251-4999 of protein 1 influenzae (strain SEQ ID NO: 497 PAK 12085) 50 5.00E−07 AcpP Pasteurella NP_246856.1 nt. 2-136 of multocida SEQ ID NO: 50 501 7.00E−50 conserved Vibrio cholerae NP_231403.1 compliment of hypothetical protein nt. 3649-4872 of SEQ ID NO: 501 501 0 type I restriction Vibrio cholerae NP_231400.1 compliment of enzyme HsdR, nt. 1551-3440 of putative SEQ ID NO: 501 501 4.00E−13 ATP-dependent Deinococcus NP_295921.1 compliment of DNA helicase radiodurans nt. 5317-5844 of RecG-related SEQ ID NO: 501 protein 501 5.00E−11 conserved Ureaplasma NP_077868.1 compliment of hypothetical urealyticum nt. 5098-5769 of SEQ ID NO: 501 504 2.00E−44 OUTER Haemophilus Q48218 compliment of MEMBRANE influenzae nt. 4681-5019 of PROTEIN P2 AG30010 SEQ ID NO: 504 PRECURSOR (OMP P2) 507 0 SpoT Pasteurella NP_245857.1 compliment of multocida nt. 3685-5316 of SEQ ID NO: 507 51 6.00E−87 glucosamine-- Vibrio cholerae NP_230141.1 nt. 30-470 of fructose-6- SEQ ID NO: 51 phosphate aminotransferase (isomerizing) 512 2.00E−28 dipeptide transport Yersinia pestis NP_407439.1 compliment of system permease nt. 1095-1580 of protein SEQ ID NO: 512 512 3.00E−82 SapC Pasteurella NP_245850.1 compliment of multocida nt. 730-1095 of SEQ ID NO: 512 514 9.00E−06 putative integral Campylobacter NP_281236.1 compliment of membrane protein jejuni nt. 577-684 of SEQ ID NO: 514 514 3.00E−11 orf, hypothetical Escherichia coli NP_286004.1 compliment of protein O157:H7 EDL933 nt. 449-568 of SEQ ID NO: 514 518 0 putative inner Neisseria NP_284893.1 nt. 92-1927 of membrane transacylase meningitidis SEQ ID NO: 518 protein Z2491 519 4.00E−30 hypothetical protein Mesorhizobium NP_108196.1 compliment of loti nt. 2221-3159 of SEQ ID NO: 519 519 2.00E−12 conserved Listeria innocua NP_471067.1 compliment of hypothetical protein nt. 3994-5241 of SEQ ID NO: 519 519 6.00E−20 hypothetical protein Mesorhizobium NP_108198.1 compliment of loti nt. 707-1552 of SEQ ID NO: 519 519 4.00E−26 putative Salmonella NP_455526.1 compliment of bacteriophage enterica subsp. nt. 3982-5163 of protein enterica serovar SEQ ID NO: 519 Typhi 52 3.00E−94 OUTER Haemophilus Q48218 nt. 45-788 of MEMBRANE influenzae SEQ ID NO: 52 PROTEIN P2 PRECURSOR (OMP P2) 520 0 excision nuclease Escherichia coli NP_418482.1 compliment of subunit A K12 nt. 6309-7745 of SEQ ID NO: 520 521 5.00E−08 zinc/manganese Rickettsia conorii NP_359651.1 nt. 2236-2652 of ABC transporter SEQ ID NO: 521 substrate binding protein 521 1.00E−140 unknown Pasteurella NP_245865.1| nt. 338-1390 of multocida SEQ ID NO: 521 521 1.00E−86 ORF_f432 Escherichia coli AAB40463.1 nt. 203-1390 of SEQ ID NO: 521 522 3.00E−22 unknown Pasteurella NP_246093.1 nt. 670-885 of multocida SEQ ID NO: 522 526 5.00E−33 exodeoxyribonuclease Yersinia pestis NP_404635.1 nt. 5582-6202 of V alpha chain SEQ ID NO: 526 526 1.00E−62 exodeoxyribonuclease Vibrio cholerae NP_231950.1 nt. 5675-6193 of V, 67 kDa SEQ ID NO: 526 subunit 527 1.00E−147 unknown Pasteurella NP_245980.1 nt. 4283-5203 of multocida SEQ ID NO: 527 527 0 Mfd Pasteurella NP_245978.1 nt. 7545-8759 of multocida SEQ ID NO: 527 527 0 transcription-repair Salmonella NP_455708.1 nt. 7611-8762 of coupling factor enterica subsp. SEQ ID NO: 527 (TrcF) enterica serovar Typhi 527 0 PROBABLE Ralstonia NP_519763.1 nt. 7611-8870 of TRANSCRIPTION- solanacearum SEQ ID NO: 527 REPAIR COUPLING FACTOR PROTEIN 528 1.00E−48 undecaprenyl Chlamydia NP_297109.1 nt. 2918-3712 of pyrophosphate muridarum SEQ ID NO: 528 synthetase 528 0 leucyl-tRNA Vibrio cholerae NP_230603.1 compliment of synthetase nt. 180-2822 of SEQ ID NO: 528 529 1.00E−104 DNA PRIMASE Legionella P71481 compliment of pneumophila nt. 3316-3960 of SEQ ID NO: 529 534 9.00E−29 putative integrase Salmonella NP_461690.1 nt. 4668-5009 of typhimurium LT2 SEQ ID NO: 534 534 6.00E−18 hypothetical protein Neisseria NP_283002.1 compliment of NMA0153 meningitidis nt. 5933-6337 of Z2491 SEQ ID NO: 534 534 2.00E−23 hypothetical protein Deinococcus NP_294868.1 nt. 6908-7654 of radiodurans SEQ ID NO: 534 534 1.00E−88 prophage CP4-57 Escherichia coli NP_417111.1 nt. 5057-5875 of integrase K12 SEQ ID NO: 534 535 1.00E−115 phosphate Buchnera sp. NP_240007.1 nt. 3385-4596 of acetyltransferase APS SEQ ID NO: 535 536 3.00E−35 cobalt membrane Actinobacillus AAD49727.1 compliment of transport protein pleuropneumoniae nt. 3531-4136 of CbiQ SEQ ID NO: 536 536 6.00E−37 unknown Pasteurella NP_245305.1 compliment of multocida nt. 6478-6921 of SEQ ID NO: 536 539 2.00E−26 Orf122 Chlorobium AAG12204.1 compliment of tepidum nt. 1778-2008 of SEQ ID NO: 539 540 1.00E−77 heat shock protein Neisseria NP_273864.1 compliment of HtpX meningitidis nt. 2567-3481 of MC58 SEQ ID NO: 540 541 0 IleS Pasteurella NP_246601.1 nt. 3167-4549 of multocida SEQ ID NO: 541 545 2.00E−09 DNA-BINDING Pectobacterium Q47588 nt. 3816-3977 of PROTEIN RDGB carotovorum SEQ ID NO: 545 545 2.00E−11 putative Sinorhizobium NP_437741.1 compliment of transposase meliloti nt. 2786-3019 of SEQ ID NO: 544 545 2.00E−07 Hypothetical 42.5 kd Escherichia coli BAA77933.1 compliment of protein in thrW- nt. 2614-2811 of argF intergenic SEQ ID NO: 545 region 545 4.00E−18 putative IS element Salmonella NP_454711.1 nt. 1955-2230 of transposase enterica subsp. SEQ ID NO: 545 enterica serovar Typhi 546 0 HEME/HEMOPEXIN- Haemophilus P45354 nt. 5551-7809 of BINDING influenzae SEQ ID NO: 546 PROTEIN 546 0 HEME/HEMOPEXIN Haemophilus P45356 nt. 3842-5536 of UTILIZATION influenzae SEQ ID NO: 546 PROTEIN B 546 0 HEME/HEMOPEXIN Haemophilus P45357 nt. 1638-3176 of UTILIZATION influenzae SEQ ID NO: 546 PROTEIN C 546 2.00E−12 HasR Pasteurella NP_246561.1 nt. 3149-3763 of multocida SEQ ID NO: 546 549 0 unknown Pasteurella NP_246821.1 nt. 2526-3512 of multocida SEQ ID NO: 549 549 1.00E−121 putative membrane Yersinia pestis NP_404859.1 nt. 605-1108 of protein SEQ ID NO: 549 549 0 unknown Pasteurella NP_246822.1 nt. 1122-1664 of multocida SEQ ID NO: 549 551 1.00E−157 type I restriction- Xylella fastidiosa NP_300016.1 compliment of modification 9a5c nt. 7396-8322 of system SEQ ID NO: 551 endonuclease 552 1.00E−100 valyl-tRNA Deinococcus NP_293872.1 compliment of synthetase radiodurans nt. 6691-8688 of SEQ ID NO: 552 552 0 VALYL-TRNA Haemophilus P36432 compliment of SYNTHETASE parainfluenzae nt. 5850-6647 of SEQ ID NO: 552 553 0 DNA-directed RNA Vibrio cholerae NP_229982.1 nt. 2668-6699 of polymerase, beta SEQ ID NO: 553 subunit 554 0 iron utilization Haemophilus T10887 nt. 991-2508 of protein B influenzae SEQ ID NO: 554 559 1.00E−100 PREPROTEIN Bacillus firmus P96313 nt. 3420-4472 of TRANSLOCASE SEQ ID NO: 559 SECA SUBUNIT 56 2.00E−23 RpL30 Pasteurella NP_246336.1 compliment of multocida nt. 656-832 of SEQ ID NO: 56 56 9.00E−13 RpS5 Pasteurella NP_246337.1 compliment of multocida nt. 843-1064 of SEQ ID NO: 56 560 1.00E−157 Na+/H+ antiporter Vibrio cholerae NP_231535.1 2 compliment of nt. 279-2989 of SEQ ID NO: 560 562 1.00E−72 putative biotin Yersinia pestis NP_404419.1 nt. 7862-8878 of sulfoxide reductase 2 SEQ ID NO: 562 562 1.00E−125 restriction Neisseria CAA09003.1 nt. 2-985 of modification meningitidis SEQ ID NO: 562 system-R protein 563 0 IMMUNOGLOBULIN Haemophilus P45384 compliment of A1 PROTEASE influenzae HK715 nt. 4127-9508 of SEQ ID NO: 563 563 0 3- Schizosaccharomyces O14289 nt. 1980-3983 of ISOPROPYLMALATE pombe SEQ ID NO: 563 DEHYDRATASE (IPMI) 564 2.00E−79 orf32 Haemophilus NP_536839.1 nt. 6241-6831 of phage HP2 SEQ ID NO: 564 564 7.00E−33 probable variable Salmonella NP_457882.1 nt. 3707-4177 of tail fibre protein enterica subsp. SEQ ID NO: 564 enterica serovar Typhi 564 2.00E−14 M protein Enterobacteria NP_052264.1 nt. 1905-2213 of phage 186 SEQ ID NO: 564 564 4.00E−44 similar to tail fiber Salmonella NP_461635.1 nt. 3171-3692 of protein (gpH) in typhimurium LT2, SEQ ID NO: 564 phage P2 Fels-2 prophage 564 2.00E−85 gpJ Enterobacteria NP_046773.1 nt. 2267-3166 of phage P2 SEQ ID NO: 564 564 1.00E−24 hypothetical protein Neisseria NP_284534.1 nt. 6852-7334 of meningitidis SEQ ID NO: 564 Z2491 564 4.00E−26 gpV Enterobacteria NP_046771.1 nt. 1337-1912 of phage P2 SEQ ID NO: 564 564 2.00E−47 similar to Escherichia coli BAA16182.1 nt. 11383-11961 [SwissProt P44255 of SEQ ID NO: 564 564 2.00E−51 hypothetical protein Neisseria NP_284066.1 nt. 10452-11180 NMA1315 meningitidis of SEQ ID NO: Z2491 564 564 0 orf31 Haemophilus NP_043502.1 nt. 4160-6226 of phage HP1 SEQ ID NO: 564 564 2.00E−09 rep Haemophilus NP_536816.1 compliment of phage HP2 nt. 9986-10234 of SEQ ID NO: 564 565 2.00E−57 resolvase/integrase- Haemophilus AAL47097.1 nt. 11885-12445 like protein influenzae biotype of SEQ ID NO: aegyptius 565 565 1.00E−93 integrase Actinobacillus AAC70901.1 compliment of actinomycetemcomitans nt. 4118-4900 of SEQ ID NO: 565 565 6.00E−35 probable phage Salmonella NP_458745.1 compliment of integrase enterica subsp. nt. 4148-4990 of enterica serovar SEQ ID NO: 565 Typhi 565 1.00E−107 hypothetical protein Xylella fastidiosa NP_299042.1 compliment of 9a5c nt. 5066-6817 of SEQ ID NO: 565 566 1.00E−126 hypothetical protein Haemophilus S15287 compliment of (galE 5′ region)- influenzae nt. 10726-11607 of SEQ ID NO: 566 567 0 unknown Pasteurella NP_246387.1 nt.5343-7688 of multocida SEQ ID NO: 567 568 1.00E−151 multidrug Escherichia coli NP_311575.1 nt. 6-1403 of resistance O157:H7 SEQ ID NO: 568 membrane translocase 568 1.00E−141 YhbX/YhjW/YijP/YjdB Neisseria |NP_275002.1 compliment of family protein meningitidis nt. 11213-12634 MC58 of SEQ ID NO: 568 570 1.00E−180 hypothetical protein Haemophilus S71024 compliment of 3 (ksgA-lic2B influenzae (strain nt. 12845-13720 intergenic region) RM7004) of SEQ ID NO: 570 571 0 glycerophosphodiester Haemophilus A43576 nt. 1656-2693 of phosphodiesterase influenzae (isolate SEQ ID NO: 571 772) 571 1.00E−137 outer membrane Haemophilus A43604 nt. 6145-6909 of protein P4 influenzae SEQ ID NO: 571 precursor - Haemophilus influenzae 571 2.00E−72 CG8298 gene Drosophila AAF58597.1 nt. 3813-5339 of product [alt 1] melanogaster SEQ ID NO: 571 572 1.00E−40 hypothetical protein Chlamydia G81737 nt. 3734-4099 of TC0130 muridarum (strain SEQ ID NO: 572 Nigg) 572 5.00E−10 hypothetical protein Pyrococcus NP_142215.1 nt. 4472-4888 of horikoshii SEQ ID NO: 572 572 3.00E−11 109aa long Sulfolobus NP_377117.1 nt. 7303-7470 of hypothetical protein tokodaii SEQ ID NO: 572 572 8.00E−43 hypothetical protein Chlamydophila NP_445524.1 nt. 4289-4618 of pneumoniae SEQ ID NO: 572 AR39 572 9.00E−08 CDH1-D Gallus gallus AAL31950.1 nt. 7183-7521 of SEQ ID NO: 572 575 1.00E−173 topoisomerase B Salmonella NP_458624.1 nt. 18980-20923 enterica subsp. of SEQ ID NO: enterica serovar 575 Typhi 575 1.00E−100 DNA helicase Salmonella NP_458617.1 nt. 10399-11706 enterica subsp. of SEQ ID NO: enterica serovar 575 Typhi 65 2.00E−53 Sufl Pasteurella NP_245041.1 nt. 3-821 of multocida SEQ ID NO: 65 67 4.00E−39 putative MFS Salmonella NP_462786.1 compliment of family tranport typhimurium LT2 nt. 125-1033 of protein (1st mdule) SEQ ID NO: 67 7 4.00E−29 putative membrane Salmonella NP_458664.1 compliment of protein enterica subsp. nt. 2-559 of enterica serovar SEQ ID NO: 7 Typhi 72 2.00E−51 serine transporter Vibrio cholerae NP_230946.1 nt. 18-803 of SEQ ID NO: 72 74 3.00E−90 hypothetical 21.8K Haemophilus JH0436 compliment of protein (in locus influenzae nt. 248-766 of involved in SEQ ID NO: 74 transformation)- 77 2.00E−18 RecX protein Legionella CAC33485.1 nt. 480-920 of pneumophila SEQ ID NO: 77 82 4.00E−95 unknown Pasteurella NP_246414.1 nt. 128-955 of multocida SEQ ID NO: 82 83 2.00E−66 unknown Pasteurella NP_246777.1 nt. 5-556 of multocida SEQ ID NO: 83 83 6.00E−33 CTP SYNTHASE Helicobacter NP_223042.1 compliment of pylori J99 nt. 1027-1338 of SEQ ID NO: 83. 83 4.00E−34 CTP synthase Campylobacter NP_281249.1 compliment of jejuni nt. 1024-1275 of SEQ ID NO: 83 84 1.00E−16 REPRESSOR Bacteriophage P14819 nt. 823-1233 of PROTEIN CI phi-80 SEQ ID NO: 84 84 2.00E−05 orf, hypothetical Escherichia coli NP_415875.1 compliment of protein K12 nt. 533-700 of SEQ ID NO: 84 84 4.00E−11 orf33 bacteriophage phi NP_490633.1 compliment of CTX nt. 32-466 of SEQ ID NO: 84 85 3.00E−42 SpoT Pasteurella NP_245857.1 nt. 899-1261 of multocida SEQ ID NO: 85 90 1.00E−103 putative methylase Bacteriophage NP_108695.1 compliment of Tuc2009 nt. 478-1206 of SEQ ID NO: 90 90 4.00E−11 probable adenine Thermoplasma NP_394624.1 compliment of specific DNA acidophilum nt. 397-1140 of methyltransferase SEQ ID NO: 90

TABLE 3B Full Length Nucleotide Amino Acid Homology to Genbank Hit Identity Sequence Sequence Location in Contig Protein CpdB SEQ ID NO: SEQ ID NO: nt. 38041-36068 of NP_246953.1 686 687 SEQ ID NO: 681 (contig 14) putative membrane SEQ ID NO: SEQ ID NO: nt. 906601-908094 NP_458664.1 protein 688 689 of SEQ ID NO: 685 (contig 18) GTP-binding SEQ ID NO: SEQ ID NO: nt. 42557-40995 of NP_240245.1 protein TypA/BipA 690 691 SEQ ID NO: 683 (contig 16) outer membrane SEQ ID NO: SEQ ID NO: nt. 7000420-704187 T30852 protein A 692 693 of SEQ ID NO: 685 (contig 18) vacB protein SEQ ID NO: SEQ ID NO: nt. 39184-36836 of NP_240369.1 694 695 SEQ ID NO: 683 (contig 16) putative ABC SEQ ID NO: SEQ ID NO: nt. 59155-58370 of NP_282774.1 transport system 696 697 SEQ ID NO: 685 permease protein [ (contig 18) putative exported SEQ ID NO: SEQ ID NO: nt. 901142-902542 NP_458655.1 protein 698 699 of SEQ ID NO: 685 (contig 18) ImpA SEQ ID NO: SEQ ID NO: nt. 348187-347747 NP_245829.1 700 701 of SEQ ID NO: 685 (contig 18) TsaA SEQ ID NO: SEQ ID NO: nt. 74941-75548 of NP_245732.1 702 703 SEQ ID NO: 684 (contig 17) PROBABLE SEQ ID NO: SEQ ID NO: nt. 74436-75176 of NP_522358.1 TRANSPORT 704 705 SEQ ID NO: 685 TRANSMEMBRANE (contig 18) PROTEIN SEQ ID NO: SEQ ID NO: nt. 75160-75660 of 706 707 SEQ ID NO: 685 (contig 18) possible exported SEQ ID NO: SEQ ID NO: nt. 899618-900262 NP_458653.1 protein 708 709 of SEQ ID NO: 685 (contig 18) LICA PROTEIN SEQ ID NO: SEQ ID NO: nt. 356917-355958 P14181 710 711 of SEQ ID NO: 685 (contig 18) HEME-BINDING SEQ ID NO: SEQ ID NO: NT. 26114-27739 P33950 PROTEIN A 712 713 of SEQ ID NO: 683 (contig 16) similar to SEQ ID NO: SEQ ID NO: nt. 311610-312683 XP_068727.1 BASEMENT 714 715 of SEQ ID NO: 685 MEMBRANE- (contig 18) SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR (HSPG) CzcD SEQ ID NO: SEQ ID NO: nt. 34865-35542 of NP_246276.1 716 717 SEQ ID NO: 681 (contig 14) conserved SEQ ID NO: SEQ ID NO: nt. 194993-193977 NP_274972.1 hypothetical protein 718 719 of SEQ ID NO: 685 (contig 18) secretion protein SEQ ID NO: SEQ ID NO: nt. 203707-201857 NP_252510.1 SecD 720 721 of SEQ ID NO: 683 (contig 17) ABC transporter SEQ ID NO: SEQ ID NO: nt. 3943-5859 of AAF31030.1 protein 1 722 723 SEQ ID NO: 681 (contig 14) conserved SEQ ID NO: SEQ ID NO: nt. 331090-331749 NP_273467.1 hypothetical protein 724 725 of SEQ ID NO: 685 (contig 18) SEQ ID NO: SEQ ID NO: nt. 331938-332492 726 727 of SEQ ID NO: 685 (contig 18) SEQ ID NO: SEQ ID NO: nt. 332681-33232 728 729 of SEQ ID NO: 685 (contig 18) INVASIN SEQ ID NO: SEQ ID NO: nt. 416757-417020 P31489 PRECURSOR 730 731 of SEQ ID NO: 685 (OUTER (contig 18) MEMBRANE ADHESIN) HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 229430-232195 P45355 BINDING 732 733 of SEQ ID NO: 384 PROTEIN (contig 17) OPACITY SEQ ID NO: SEQ ID NO: nt. 375592-375879 Q05033 PROTEIN OPA66 734 735 of SEQ ID NO: 384 PRECURSOR (contig 17) Hemoglobin- SEQ ID NO: SEQ ID NO: nt. 45709-42566 of Q48153 haptoglobin binding 736 737 SEQ ID NO: 681 protein A (contig 14) transport protein SEQ ID NO: SEQ ID NO: nt. 134452-135222 NP_253757.1 TatC 738 739 of SEQ ID NO: 384 (contig 17) LIPOPROTEIN SEQ ID NO: SEQ ID NO: nt. 18895-20112 of P40827 NLPD 740 741 SEQ ID NO: 682 (contig 15) Hemoglobin and SEQ ID NO: SEQ ID NO: nt. 34181-31041 of Q9X442 hemoglobin- 742 743 SEQ ID NO: 682 haptoglobin binding (contig 15) protein C precursor HimA SEQ ID NO: SEQ ID NO: nt. 382795-383085 NP_245565.1 744 745 of SEQ ID NO: 685 (contig 18) transferrin-binding SEQ ID NO: SEQ ID NO: nt. 178537-175799 S70906 protein 1 746 747 of SEQ ID NO: 683 (contig 16) SapC SEQ ID NO: SEQ ID NO: nt. 197754-196867 NP_245850.1 748 749 of SEQ ID NO: 685 (contig 18) heat shock protein SEQ ID NO: SEQ ID NO: nt. 40414-41265 of NP_273864.1 HtpX 750 751 SEQ ID NO: 682 (contig 15) HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 229430-232195 P45354 BINDING 752 753 of SEQ ID NO: 684 PROTEIN (contig 17) HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 227721-229418 P45356 UTILIZATION 754 755 of SEQ ID NO: 684 PROTEIN B (contig 17) HEME/HEMOPEXIN SEQ ID NO: SEQ ID NO: nt. 225516-227645 P45357 UTILIZATION 756 757 of SEQ ID NO: 684 NP_246561.1 PROTEIN C (contig 17) iron utilization SEQ ID NO: SEQ ID NO: nt. 32076-33611 of T10887 protein B 758 759 SEQ ID NO: 684 (contig 17) PREPROTEIN SEQ ID NO: SEQ ID NO: nt. 82314-84785 of P96313 TRANSLOCASE 760 761 SEQ ID NO: 683 SECA SUBUNIT (contig 16) IMMUNOGLOBULIN SEQ ID NO: SEQ ID NO: nt. 171647-166263 P45384 A1 PROTEASE 762 763 of SEQ ID NO: 683 (contig 16) multidrug SEQ ID NO: SEQ ID NO: nt. 74524-72992 of NP_311575.1 resistance 764 765 SEQ ID NO: 683 membrane (contig 16) translocase YhbX/YhjW/YijP/YjdB SEQ ID NO: SEQ ID NO: nt. 61734-63200 of NP_275002.1 family protein 766 767 SEQ ID NO: 683 (contig 16) putative membrane SEQ ID NO: SEQ ID NO: nt. 906601-908094 NP_458664.1 protein 768 769 of SEQ ID NO: 685 (contig 18) putative membrane SEQ ID NO: SEQ ID NO: nt. 16185-17942 of NP_404859.1 protein 770 771 SEQ ID NO: 683 (contig)

Example 3 Construction of the NTHi Promoter Trap Library

To identify potential virulence determinants of NTHi, bacterial gene expression was monitored by differential fluorescence induction (DFI) during early disease progression in one specific anatomical niche of a chinchilla model of NTHi-induced otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene using a promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1 isolated from Actinobacillus pleuropneumoniae, is an A. pleuropneumoniae-Escherichia coli shuttle vector. This plasmid contains the origin of replication from A. pleuropneumoniae, the lacZα gene from pUC19 and the kanamycin resistance gene from Tn903. (West et al., Genes, 160: 81-86, 1995).

The promoter trap vector was constructed by cloning the GTP mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to form pRSM2167. This mutant GTP gene contains two amino acid changes, S65G and S72A, that enhance fluorescence emission when excited at 488 nm. This mutant also has high solubility and fast kinetics of chromophore formation (Cormack et al., Gene, 173: 33-38, 1906). This plasmid was transformed by electroporation into NTHi strain 86-028NP, generating the parent-plasmid strain 86-028NP/pRSM2169.

Random genomic DNA fragments (described in Example 1) were prepared for ligation into the promoter probe vector. Genomic DNA was isolated from strain 86-028NP using the Puregene DNA isolation kit (Gentra Systems, Minneapolis. MN) according to the manufacturer's protocol. Due to restriction barriers, it was necessary to isolate the plasmid DNA and use this for the library generation. The isolated DNA was partially digested with Sau3AI (NEB, Beverly, Mass.; 0.25 units/μg DNA) for 1 hour at 37° C., separated by gel electrophoresis and DNA fragments 0.5-1.5 kb in size were recovered using the Qiagen gel extraction kit. For vector preparation, pRSM2167 was isolated from an overnight culture using the Wizard Plus Maxiprep DNA purification system (Promega, Madison WD according to the manufacturer's protocol.

Plasmid DNA was linearized by BamHI digestion and 5′ phosphate groups removed by treatment with calf intestinal alkaline phosphatase (CLAP; GibcoBRL Life Technologies). Genomic DNA fragments were ligated with the linearized, phosphatase-treated vector and electroporated into competent NTHi strain 86-028NP prepared for electroporation according to a modified protocol (Mitchell et al., Nucleic Acids Res., 19: 3625-3628, 1991). When plasmid DNA was electroporated back into NTHi strain 86-028NP, transformation efficiency was improved by one-thousand fold. Briefly, cells were grown to an OD₆₀₀=0.3 in sBHI (brain heart infusion) broth at 37° C., 220 rpm. Cells were chilled on ice for 30 minutes and subsequently washed with an equal volume of 0.5×SG (1×SG: 15% glycerol, 272 mM sucrose) at 4° C. Washes were repeated a total of three times. Subsequently, the cells were diluted in 1×SG to a 100× concentrated volume. The cells were electroporated using the BioRad Gene Pulser II set at 200 ohms, 2.5 kV and 25 μF and then diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37° C., 5% CO₂ and plated on chocolate agar for overnight growth of transformants.

Transformants were selected and frozen in pools of 1000 clones in skim milk containing 20% glycerol (vol/vol). A 68,000 member gfp promoter probe library was generated. Using the probability calculation of Clarke and Carbon (Cell. 9: 91-99, 1976), to achieve a 99% probability of having a given DNA sequence represented in a library of 300 bp fragments of strain 86-028NP DNA (1.8×10⁶ bp/genome), a library of 27.629 clones was needed. Therefore the present library represents 2.5 fold coverage of the 86-028NP genome.

In order to assess the quality of the library, fifty clones were selected at random, grown overnight on chocolate agar and the plasmids were isolated and insert DNA sequenced. A majority (64%) of the selected clones had insert sizes ranging between 200 and 500 bp while 32% exceeded 500 bp. The majority of inserts showed homology to unique H. influenzae strain Rd open reading frames (ORFs), and 15 clones had sequence unique to strain 86-028NP DNA. Of those clones with homology to strain Rd, 60% were in the correct orientation, 36% of which contained sequence upstream an ORF. Although a majority of clones had an insert size less than 500 bp, no correlation was found between small insert size and increased GFP expression. In fact four clones exhibited slight to moderate fluorescence in vitro, 3 of which had insert sizes between 200-500 base pairs and one had an insert that was greater than 700 base pairs.

A fraction of the library (approximately 1000 clones) was grown on chocolate agar, harvested in PBS and analyzed by flow cytometry for GFP fluorescence. Compared to strain 86-028NP/pRSM2169 that contains the promoter trap vector without insert DNA, the pool of library clones displays an increased fluorescence intensity. Thus, the library contains clones with promoters at varying levels of activity.

Example 4 Analysis of 86-028NP Derivatives Expressing GFP

In order to establish the FACS parameters necessary to identify and sort gfp-expressing bacteria, a panel of isolates demonstrating varying levels of gfp expression was utilized. Background fluorescence was assessed using strain 86-028NP/pRSM2169 (negative control), therefore any observed fluorescence would be due to the lacZ promoter driving gfp expression. However, this strain does not produce detectable levels of GFP and in fact, does not demonstrate increased fluorescence when compared to the parent strain 86-028NP. A high-level gfp-expressing isolate was generated by cloning a 500 bp fragment containing the strong promoter for outer membrane protein P2 expression into SalI-BamHI digested pRSM2167. This plasmid was transformed into 86-028NP by electroporation, generating the high-level gfp expressing strain 86-028NP/pRSM2211 (highly fluorescent control). This strain demonstrated an approximate 100 fold increase in GFP fluorescence compared to strain 86-028NP/pRSM2169. An intermediate fluorescent derivative clone, 86-028NP/pKMM4B5 (intermediate fluorescent control), was isolated by FACS analysis and used both in preliminary experiments and as a control for cell sorting. The DNA fragment containing a promoter driving gfp expression in vitro is unique to strain 86-028NP, having no known homology to DNA of other organisms. This clone exhibits an approximate 10 fold increase in fluorescence compared to strain 86-028NP/pRSM2169.

The control strains were resuspended from growth on chocolate agar and labeled with cross-reactive Phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μlPBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, bacteria were resuspended in 300 μl Dulbecco's Phosphate Buffered Saline (DPBS) DPBS for FACS analysis. These control preparations were used to set the appropriate size and fluorescence gates using a Coulter Epics Elite flow cytometer (Coulter Corp.) equipped with an argon laser emitting at 488 nm. Bacteria were gated for size based on log forward angle and side scatter detection and for sorting by FITC/PE labeling of bacteria. Sorted cells were collected into cold sBHI and plated on chocolate agar. After overnight growth, cells were collected for a secondary round of infection or were individually selected and grown overnight, screened by individual clone for fluorescence when grown in vitro, and frozen in skim milk containing 20% (vol/vol) glycerol prior to plasmid isolation and sequencing of insert DNA. Sorting efficiency of control strains was confirmed using a Coulter EPICS flow cytometer (Coulter Corp.).

Many plasmids were segregated rapidly in vitro in the absence of antibiotic selection. Thus, in order to assess whether the promoter trap vector used here was prone to this event, a single colony of strain 86-028NP/pRSM2211 (highly fluorescent control) was isolated on chocolate agar and passaged 20 times in the absence of antibiotic selection. No significant decrease in fluorescence intensity was observed when compared to bacteria grown in the presence of antibiotic. In addition, the plasmid is maintained in the absence of antibiotic selection in vivo. Similar bacterial counts were observed when bacteria-containing middle ear fluids collected from a chinchilla were plated on chocolate agar with or without kanamycin. These data demonstrate that the promoter trap vector was stably maintained in the absence of antibiotic selection.

In addition to problems with plasmid stability, early studies on the use of GFP as a reporter to study host-pathogen interactions demonstrated that GFP could be continuously synthesized as a cytoplasmic protein with low toxicity, having minimal effects on the bacterial cell-surface dynamics (Chalfie et al., Science, 263: 802-805, 1994). The construction of a high level gfp-expressing derivative allowed the assessment of the GFP toxicity on NTHi. Growth curves of both the wild-type strain (86-028NP) and the high GFP producing strain 86-028NP pRSM2211 were compared when grown under similar conditions. The growth rates were similar, indicating that GFP expression was not toxic to the cells.

The 86-028NP gfp-expressing derivatives were used to define the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169 was mixed with the intermediate gfp-ex pressing derivative, strain 86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo environment that is expected to contain a small percentage of gfp-expressing clones relative to the total bacterial population. This mixture was subjected to FACS analysis, collecting the 1.8% most fluorescent population and the 52% least fluorescent population. Flow cytometric analysis of the sorted populations revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the bacterial population, a phenomenon that was not observed when sorting on the negative population. Subsequent rounds of sorting would be expected to further enrich for this intermediate fluorescent population. The inability to decrease the amount of fluorescent bacteria in the negative sort was attributed to the size of the gate set for negative sorting. GFP-negative cells were enriched by gating on the 10% least fluorescent population.

Example 5 Direct Labeling of Bacteria from Middle Ear Fluids

A similar strategy (as described in Example 5) was applied to sort fluorescent clones from effusions obtained from the chinchilla middle ear during AOM. Our ability to use differential fluorescence induction (DFI) in vivo was dependent upon our ability to sort gfp-expressing bacteria from non-fluorescent bacteria, fluorescent and non-fluorescent cellular debris, and eukaryotic cells.

Healthy adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either otoscopy or tympanometry were used to screen the library for promoter activity in vivo. Two pools of the NTHi/pRSM2169 library (1000 clones each) were grown overnight on chocolate agar containing kanamycin. The library was combined and diluted in cold 10 mM sterile PBS to 3.3×10⁶ CFU/ml and 300 μl (1.0×10⁶ CPU; 500 CFU/clone) was used to inoculate the left and the right chinchilla transbullar cavity (2000 clones/ear). OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. The bacteria multiplied in the middle ear cavity, reaching a concentration 500 times the inoculum dose by 48 hours as expected (Bakaletz et al., Inject. Immunity 67; 2746-62, 1999). This bacterial adaptation to the host environment results in an inflammatory response, indicated by erythema, vessel dilation and bulging of the tympanic membrane, infiltration of polymorphonuclear cells (PMN's), and accumulation of fluid in the middle ear cavity as observed by otoscopy and microscopic examination of recovered effusions. Twenty-four and 48 hours later, middle ear fluids were retrieved by epitympanic tap, and prepared for FACS.

It is important to note that this analysis was limited to those bacteria recoverable in the middle ear fluid. In some cases it was necessary to lavage the middle ear cavity to collect the bacteria for FACS analysis. Thus, this analysis includes genes up-regulated when NTHi are loosely adherent to mucosae. NTHi has been observed to form a biofilm in the middle ear cavity in a chinchilla model of OM (Erhlich et al., JAMA, 287; 1710-5, 2002). Since the protocols described herein select for clones recovered from the planktonic population, it is not expected to recover those clones in which genes are up-regulated when the bacteria are associated with mucosal biofilms. Homogenization of middle ear mucosae and subsequent bacterial cell isolation however, would enable us to recover these clones. It is also possible that some GFP-expressing clones were recovered in the effusion, yet were adherent to eukaryotic cells present in the effusion as exfoliated cells, or in aggregates. These bacteria are difficult to recover from the effusion without compromising the sorting efficiency. Therefore the middle ear fluids were treated with a mucolytic agent, then centrifuged to remove large aggregates and eukaryotic cells and prior to labeling.

Chinchilla middle ear fluids were diluted, if necessary, to 250 μl with sterile saline. An equal volume of N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5 minutes at room temperature as a mucolytic agent (Miyamoto and Bakaletz, Microb. Pathog., 21: 343-356 1996). Fluids were centrifuged (300×g, 5 min) to remove cellular debris, red blood cells and inflammatory cells, and supernatants containing bacteria were transferred to a fresh tube. Bacteria were incubated with chinchilla antiserum (1:50 dilution) directed against a whole OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes at 4° C. pelleted by centrifugation (2000×g, 5 min) and washed twice with cold DPBS containing 0.05% bovine serum albumin. Bacteria were subsequently labeled with cross-reactive phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, cells were resuspended in 300 μl DPBS for FACS analysis.

Example 6 Identification of Promoters Induced In Vivo in Acute Otitis Media

H. influenzae 86-028NP transformed with the promoter trap library was grown overnight on chocolate agar. To select against those clones containing promoters that expressed gfp in vitro, the library was subjected to one round of FACS analysis (as described in Example 6), collecting only those clones expressing low-level amounts of GFP. These clones were pooled and used to inoculate the chinchilla middle ear transbullarly. Following 24 and 48 hours of infection, bacteria-containing effusions were removed by epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled antibody and subjected to FACS analysis by gating on fluorescently tagged bacteria but sorting for those that were also expressing. These clones were used to reinfect animals for further enrichment. Following the final round of sorting, single colony isolates were screened in vitro for lack of fluorescence.

Those clones isolated by FACS analysis (positive for GFP fluorescence in vivo), which did not emit fluorescence in vitro were prepared for plasmid isolation and identification of insert DNA sequence. These clones were grown overnight on chocolate agar plates containing kanamycin and prepared for plasmid isolation using the Qiaprep Miniprep Kit (Qiagen) according to the manufacturer's protocol. Plasmid insert DNA was sequenced using the primer 5′-TGCCCATTAACATCACCATCTA-3*(SEQ ID NO: 588) that is complementary to the gfpmut3 gene and downstream of the insert DNA. Sequencing reactions were performed using the ABI prism BigDye® terminator cycle sequencing ready reaction kit (Applied Biosystems) according to manufacturer's protocol using a GeneAmp PCR System 9700 (Applied Biosystems). The sequences were then purified by passage through sephadex G-50 in a 96-well multiscreen HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100 DNA analyzer (Applied Biosystems).

Insert sequences were compared to the complete annotated sequence of H. influenzae strain Rd. Those inserts with no nucleotide homology to strain Rd were subsequently analyzed using the BLASTN and BLASTX algorithms. Further sequence analysis was performed with DNA STAR (Madison, Wise). Inserts in the correct orientation and containing sequence 5′ to a predicted ORF contained a putative promoter that was preferentially active when the NTHi bacteria were in the chinchilla middle ear.

Fifty-two clones with putative promoters that were regulated in vivo were isolated. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear and these genes are summarized in Table 4A below. The in vivo-regulated promoters driving expression of genes are predicted to be involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function.

In order to confirm the induction of putative promoter candidates in vivo, the relative amount of messenger RNA expression was compared when NTHi strain 86-028NP was grown in vitro to mid-log phase or in vivo for 48 hours. The RNA was isolated using TRIzol LS reagent (Gibco Life Technologies) according to the manufacturer's protocol. DNA was removed from the RNA preparation using DNA-free kit (Ambion) according to the manufacturer's protocol. DNase I treated RNA samples were purified by passage through a Qiagen RNeasy column. RNA purity and integrity was assessed by 260/280 nm spectrophotometer readings and on the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.

In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Real-time quantitative RT-PCR using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription levels according to the manufacture's instructions. Briefly, using primers generated to an open reading frame downstream of the putative in vivo-induced promoters identified by FACS analysis, gene-specific mRNA was reverse transcribed and amplified by RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems). The amount of product was calculated using a standard curve generated to known amounts of bacterial genomic DNA (10²-10⁷ genomic copies DNA) by amplifying a fragment of the gyrase (gyr) gene. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparation (-RT control) as well as the absence of primer dimers in control samples lacking template RNA. In addition, RT-PCR products were analyzed by gel electrophoresis and, in all cases, a single product was observed at the appropriate base pair size. Amounts of bacterial RNA between samples were normalized relative to gyr expression, shown to be constitutively expressed under various growth conditions that we tested in vitro. Known amounts of bacterial genomic DNA (10²-10⁷ genomic copies DNA) were used to generate a standard curve for RT-PCR quantitation by amplifying a fragment of the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro under various growth conditions and was therefore used to normalize total bacterial RNA levels between samples. Relative gene expression in vivo was compared to that of gene expression in vitro and data expressed as fold-increase are summarized in Table 4A.

The 8-fold sequencing of the NTHi genome identified the full length open reading frames for the majority of genes listed in Table 4A. Table 4B provides the full length nucleotide sequence within the NTHi genome and the corresponding amino acid sequence. The fold induction of the gene due to environmental cues present in the chinchilla middle ear and the product or function of the gene are repeated in Fable 4B for convenience.

TABLE 4A SEQ Gene or ID GenBank Fold Category ORF NO: Protein ID Induction Product or Function Amino acid hisB 589 NP_438632 2.9 Histidine biosynthesis metabolism bifunctional protein Lipoprotein lppB 590 NP_438862.1 2.6 Lipoprotein B homologue Membrane transport sapA 591 NP_439780.1 2.8 Peptide ABC transporter; periplasmic SapA precursor lolA 592 NP_439736.1 2.4 Outer membrane lipoproteins carrier protein precursor rbsC 593 NP_438661.1 5.1 Ribose transport system permease protein Purine synthesis purE 594 NP_439757.1 51.7 Phosphoribosylaminoimidazole carboxylase catalytic subunit; PurE Biosynthetic and ribB 595 NP_438923.1 8.3 3,4-dihydroxy-2-butanone 4- metabolic functions phosphate synthase; riboflavin biosynthesis arcB 596 NP_438753.1 10 Ornithine carbamolytransferase; arginine degradation uxuA 597 NP_438228.1 3.1 Mannonate dehydratase; production of glyceraldehyde 3-phosphate dsbB 598 NP_438589.1 2.6 Disulfide oxidoreductase; disulfide bond formation protein B ureH 599 NP_438693.1 3.9 Urease accessory protein licC 600 NP_439688.1 2.3 Phosphocholine (ChoP) cytidylyltransferase HI1647 601 NP_439789.1 2.0 Putative pyridoxin biosynthesis protein; singlet oxygen resistance protein DNA replication, ispZ 602 P43810 2.5 Probable intracellular repair septation protein radC 603 NP_439113.1 2.1 DNA repair protein mukF 604 P45185 2.0 MukF protein homologue; remodeling of nucleiod structure Gene regulation glpR 605 NP_438777.1, 2.8 Glycerol-3-phosphate regulon NP_439170.1 repressor ihfB 606 P43724 2.5 Integration host factor beta subunit argR 607 NP_439365.1 2.7 Arginine repressor cspD 608 NP_439584.1 2.1 Cold shock like protein; stress response protein Hypothetical or HI0094 609 NP_438267.1 8.3 Hypothetical protein unknown HI1163 610 NP_439321.1 2.3 Conserved hypothetical proteins protein; putative oxidase HI1063 611 NP_439221.1 2.7 Hypothetical protein HI0665 612 NP_438824.1 2.8 Hypothetical protein HI1292 613 NP_439444.1 2.6 Hypothetical protein HI1064 614 NP_439222.1 2.6 Hypothetical protein

TABLE 4B Gene Full Length Amino Acid Fold Product or Category or ORF Nucleotide Sequence Sequence Location in Contig Induction Function Amino hisB SEQ ID NO: SEQ ID NO: nt. 68378-67290 2.9 Histidine acid 615 616 of SEQ ID NO: 680 biosynthesis metabolism (contig 13) bifunctional protein Membrane sapA SEQ ID NO: SEQ ID NO: nt. 200403-198709 2.8 Peptide ABC transport 617 618 of SEQ ID NO: 685 transporter; (contig 18) periplasmic SapA precursor rbsC SEQ ID NO: SEQ ID NO: nt. 42773-41802 5.1 Ribose transport 619 620 of SEQ ID NO: 680 system permease (contig 13) protein Purine purE SEQ ID NO: SEQ ID NO: nt. 219625-219131 51.7 Phosphoribosylamino- synthesis 621 622 of SEQ ID NO: 685 imidazole (contig 18) carboxylase catalytic subunit; PurE Biosynthetic ribB SEQ ID NO: SEQ ID NO: nt. 131537-132184 8.3 3,4-dihydroxy-2- and 623 624 of SEQ ID NO: 682 butanone 4- metabolic (contig 15) phosphate synthase; functions riboflavin biosynthesis arcB SEQ ID NO: SEQ ID NO: nt. 49710-48706 10 Ornithine 625 626 of SEQ ID NO: 681 carbamolytransferase; (contig 14) arginine degradation uxuA SEQ ID NO: SEQ ID NO: nt. 840671-841855 3.1 Mannonate 627 628 of SEQ ID NO: 685 dehydratase; (contig 18) production of glyceraldehyde 3- phosphate dsbB SEQ ID NO: SEQ ID NO: nt. 388050-388583 2.6 Disulfide 629 630 of SEQ ID NO: 384 oxidoreductase; (contig 17) disulfide bond formation protein B ureH SEQ ID NO: SEQ ID NO: nt. 4452-5267 3.9 Urease accessory 631 632 of SEQ ID NO: 680 protein (contig 13) licC SEQ ID NO: SEQ ID NO: nt. 355083-354382 2.3 Phosphocholine 633 634 of SEQ ID NO: 385 (ChoP) (contig 18) cytidylyltransferase H11647 SEQ ID NO: SEQ ID NO: nt. 664017-664892 2.0 Putative pyridoxin 635 636 of SEQ ID NO: 685 biosynthesis protein; (contig 18) singlet oxygen resistance protein DNA ispZ SEQ ID NO: SEQ ID NO: nt. 4512-5069 2.5 Probable replication, 637 638 of SEQ ID NO: 683 intracellular repair (contig 16) septation protein radC SEQ ID NO: SEQ ID NO: nt. 132695-132030 2.1 DNA repair protein 639 640 of SEQ ID NO: 683 (contig 16) mukF SEQ ID NO: SEQ ID NO: nt. 504549-503215 2.0 MukF protein 641 642 of SEQ ID NO: 685 homologue; (contig 18) remodeling of nucleiod structure Gene glpR SEQ ID NO: SEQ ID NO: nt. 72716-73483 2.8 Glycerol-3- regulation 643 644 of SEQ ID NO: 682 phosphate regulon (contig 15) repressor ihfB SEQ ID NO: SEQ ID NO: nt. 661004-660720 2.5 Integration host 645 646 of SEQ ID NO: 685 factor beta subunit (contig 18) argR SEQ ID NO: SEQ ID NO: nt. 178540-178085 2.7 Arginine repressor 647 648 of SEQ ID NO: 685 (contig 18) cspD SEQ ID NO: SEQ ID NO: nt. 435310-435528 2.1 Cold shock like 649 650 of SEQ ID NO: 685 protein; stress (contig 18) response protein Hypothetical H11163 SEQ ID NO: SEQ ID NO: nt. 137202-134119 2.3 Conserved or unknown 651 652 of SEQ ID NO: 685 hypothetical protein; proteins (contig 18) putative oxidase H11063 SEQ ID NO: SEQ ID NO: nt. 35158-34937 2.7 Hypothetical protein 653 654 of SEQ ID NO: 685 (contig 18) H10665 SEQ ID NO: SEQ ID NO: nt. 17949-18980 2.8 Hypothetical protein 655 656 of SEQ ID NO: 679 (contig 12) H11292 SEQ ID NO: SEQ ID NO: nt. 555002-555799 2.6 Hypothetical protein 657 658 of SEQ ID NO: 685 (contig 18)

Example 7 Identification of Virulence-Associated Genes

In many bacterial species, a subset of virulence-associated genes is regulated by errors in replication of short repeats. These repeats may be 5′ to a gene or in the coding sequence, and their presence is an indication of controlled expression of the gene, which indicates association with virulence. Addition or deletion of a repeat results in the expression or of lack of expression of the particular virulence determinant.

The NTHi H. influenzae strain 86-028SNP contig set was queried for short oligonucleotide repeats. The region surrounding the repeats was analyzed to identify the gene(s) associated with the repeat. Table 5 lists the identified repeats and the ORE (identified by BLAST) associated with each repeat.

Further sequence analysis has identified the full length nucleotide sequence of the virulence-associated genes and the corresponding amino acid sequences encoded by the ORF. The derived ammo acid sequences are highly homologous to the listed Genbank sequence.

TABLE 5 Location in Location in Full Length Amino 3-fold 8-fold Nucleotide Acid Genebank Repeat Contigs Contigs Sequence Sequence Accession No. SEQ ID 115 nt. 484533-483643 SEQ ID SEQ ID NP_439538.1 NO: 581 nt. 473-540 of NO: 659 NO: 660 of SEQ ID SEQ ID NO: 685 NO: 115 (contig 18) SEQ ID 377 nt. 416274-414910 SEQ ID SEQ ID P45217 NO: 582 nt. 546-597 of NO: 661 NO: 662 of SEQ ID NO: SEQ ID 685 (contig NO: 337 18) SEQ ID 505 nt. 414500-416614 SEQ ID SEQ ID AAK76425 NO: 583 nt. 310-393 of NO: 663 NO: 664 of SEQ ID NO: SEQ ID 684 (contig NO: 505 17) SEQ ID 508 nt. 506516-507913 SEQ ID SEQ ID NP_439520 NO: 584 nt. 2079-2120 of NO: 665 NO: 666 of SEQ ID NO: SEQ ID 685 (contig NO: 508 18) SEQ ID 518 nt. 354274-352406 SEQ ID SEQ ID NP_284893 NO: 585 nt. 758-789 of NO: 667 NO: 668 of SEQ ID NO: SEQ ID 684 (contig NO: 518 17) SEQ ID 543 nt. 347864-243236 SEQ ID SEQ ID AAA20524 NO: 586 nt. 1814-196 of NO: 669 NO: 670 of SEQ ID NO: SEQ ID 685 (contig NO: 543 18) SEQ ID 543 nt. 699709-704187 SEQ ID SEQ ID AAD56660 NO: 586 nt. 1814-196 of NO: 671 NO: 672 of SEQ ID NO: SEQ ID 685 (contig NO: 543 18) SEQ ID 567 nt. 85546-84689 SEQ ID SEQ ID ZP_00053190 NO: 587 nt. 13309-13320 of NO: 673 NO: 674 of SEQ ID NO: SEQ ID 681 (contig NO: 567 14)

Example 8 Identification of Unique NTHi Gene Sequences

Genes associated with NTHi virulence were also identified by comparing the level of expression of the gene when the NTHi bacterium was infecting a tissue verses the level of expression of the same gene when the NTHi was grown on artificial laboratory media. These novel genes were identified using the promoter trap techniques described above in Examples 4-6, and subsequently comparisons with the known Rd genome demonstrated these genes are unique to NTHi strain 86-028SNP.

The DNA sequence identified using this screening procedure are set forth as SEQ ID NOS: 577-580. These sequences did not contain genes or gene fragments that have homologues in the H. influenzae Rd. genome sequence. Even though these are completely novel sequences, due to their expression level during NTHi infection in the chinchilla middle ear, it is likely that expression of these genes are involved in NTHi virulence. 

What is claimed is:
 1. A method for detecting NTHi bacteria in a biological sample comprising (a) contacting an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 633 with polynucleotides from a biological sample under stringent hybridization conditions, wherein the hybridization conditions comprise washing with 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. , and (b) detecting hybridization of the polynucleotide within the sample, wherein hybridization indicates the presence of NTHi bacteria in the biological sample.
 2. The method of claim 1 wherein the biological sample is selected from the group consisting of serum, sputum, ear fluid, blood, urine, lymphatic fluid, and cerebrospinal fluid.
 3. A vector comprising an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:
 633. 4. A vector, wherein an isolated polynucleotide comprising the nucleotide of SEQ ID NO: 663 is operatively linked to an expression control sequence.
 5. An isolated cell comprising the vector of claim
 3. 6. A host cell comprising the vector of claim
 4. 